Soluble Mediator

ABSTRACT

The present disclosure relates to a soluble CD52 glycoprotein and its use in treating diseases regulated by effector T-cells, for example autoimmune diseases such as type 1 diabetes. The present disclosure also relates to fusion proteins comprising the soluble glycoprotein, to cells expressing high levels of CD52, and to diagnostic methods based on the detection of CD52 expression levels in a subject.

FIELD OF THE INVENTION

The present disclosure generally relates to cell populations and solublemediators capable of suppressing T-cell activation, and to the use ofsuch cell populations and soluble mediators to suppress T-cellactivation, such as in the treatment of diseases or conditions mediatedby effector T-cell function. The disclosure also relates to methods ofdetecting the presence of a marker in a subject, which marker isindicative of the subject's susceptibility to diseases or conditionsmediated by effector T-cell function.

BACKGROUND OF THE INVENTION

Regulatory T-cells (Treg cells; also known as suppressor T-cells) aresubpopulations of T-cells that maintain immune homeostasis and helpavert autoimmune disease (Sakaguchi et al., 2008; Shevach, 2006; Vignaliet al., 2008). Interest in Treg cells is focused predominantly onprototypic CD4⁺ CD25⁺ Treg cells that are programmed by thetranscription factor FoxP3 (Fontenot et al., 2003; Hori et al., 2003).In resting polyspecific populations these Treg cells are characterisedin the mouse both as ‘natural’, thymus-derived and induced ‘adaptive’cells that suppress the activation, proliferation and functions of otherT-cells (Sakaguchi et al., 2008; Shevach, 2006). However, in human bloodCD4⁺ Treg cells are not as reliably distinguished by FoxP3 expression(Roncarolo and Gregori, 2008; Allan et al., 2007; Gavin et al., 2006).Thus, CD4⁺ T-cells with markers of either naïve or memory cells wereshown to have similar suppressor functions despite low and highexpression, respectively, of FoxP3 (Miyara et al., 2009). Other surfacemarkers of human CD4⁺ CD25⁺ FoxP3⁺ Treg cells such as decreasedexpression of the IL-7 receptor, CD127 (Liu et al., 2006; Seddiki etal., 2006), are not specific for Treg cells.

Aside from the paucity of specific cell surface markers, the mechanismsunderlying suppression by CD4⁺ CD25⁺ FoxP3⁺ Treg cells remaincontroversial. In the mouse, suppression ex vivo has been shown torequire cell-cell contact but has been attributed to multiple mechanisms(Vignali et al., 2008; Shevach, 2009; Sakaguchi et al., 2009); even lessis known about the function of similar human Treg cells. Furthermore,other types of both CD4⁺ and CD8⁺ Treg cells that differ in proposedmechanisms of suppressor function have been described in the context ofvarious tissue sites or diseases (Vignali et al., 2008).

Treg cells induced by administration of autoantigens have been shown toprotect against some autoimmune diseases in certain animal models(reviewed by von Herrath and Harrison, 2003). For example, in thenonobese diabetic (NOD) mouse model of type 1 diabetes (T1D) CD4⁺ Tregcells induced by administered pancreatic islet autoantigens such asinsulin (Bergerot et al., 1994) or glutamic acid decarboxylase 65(GAD65) (Tisch et al., 1999), or transfer of CD4⁺ Treg cells induced byproinsulin (Every et al., 2006) or a putative pancreatic islet antigen(Tang et al., 2004), have been shown to protect against autoimmunediabetes. However, in these models Treg cells have been studied inresting, polyspecific populations and not during the host's response toa particular antigen. Recently, proinsulin- and GAD65-specific humanCD4⁺ T-cell clones were generated and Treg clones were distinguished bytheir suppressor function in vitro (Dromey et al., 2011). The cellsurface membrane-anchored glycoprotein CD52 was shown to be upregulatedin these expanded CD4⁺ Treg clones. However, the mechanism of immunesuppression has not previously been characterized.

SUMMARY OF THE INVENTION

The present inventors have identified a soluble mediator of Treg cellsuppression. Accordingly, the present disclosure provides apharmaceutical composition comprising any one or more of:

i) soluble CD52 glycoprotein,

ii) a fusion protein comprising soluble CD52 glycoprotein as a firstprotein, and a second protein;

iii) a polynucleotide encoding the peptide portion of soluble CD52glycoprotein of part i) or the fusion protein of part ii);

iv) a vector comprising the polynucleotide of part iii);

v) an isolated cell comprising the polynucleotide of part iii) or thevector of part iv);

vi) an isolated CD52^(hi) cell capable of producing soluble CD52glycoprotein;

vii) an isolated cell population comprising a plurality of CD52^(hi)cells capable of producing soluble CD52 glycoprotein;

viii) cell culture medium, or a fraction thereof comprising soluble CD52glycoprotein, isolated from a cell culture comprising the cell of partvi) or the cell population of part vii); and

ix) an agent capable of increasing the level of expression of solubleCD52 glycoprotein by a cell;

and a pharmaceutically acceptable carrier.

In a preferred embodiment, the soluble CD52 glycoprotein comprises anamino acid sequence at least 60% identical to the amino acid sequence ofany one or more of GQNDTSQTSSPS (SEQ ID NO: 3), SQNATSQSSPS (SEQ ID NO:4), GQATTAASGTNKNSTSTKKTPLKS (SEQ ID NO: 5),GQNSTAVTTPANKAATTAAATTKAAATTATKTTTAVRKTPGKPPKA (SEQ ID NO: 6) orGNSTTPRMTTKKVKSATPA (SEQ ID NO:7) and a carbohydrate. More preferably,the glycoprotein comprises an amino acid sequence which is at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% identical, or is 100% identical, to any one or more of theamino acid sequences identified in SEQ ID NOs: 3, 4, 5, 6 or 7.

In one example, the glycoprotein comprises an amino acid sequence atleast 60% at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% identical, or is 100% identical tothe amino acid sequence of SEQ ID NO: 3, which represents the humansoluble CD52 fragment.

Preferably, any one or more of the soluble CD52 glycoprotein, fusionprotein, polynucleotide, vector, cell, cell population, cell culturemedium and agent is present in an amount sufficient to suppress effectorT-cell function and/or an immune response.

In a further embodiment, the soluble CD52 glycoprotein, fusion protein,polynucleotide, vector, cell, cell population, cell culture medium andagent is present in an amount sufficient such that the suppression ofthe immune response results in tolerance to at least one antigen such anautoantigen.

In another embodiment, any one or more of the soluble CD52 glycoprotein,fusion protein, cell, cell population, cell culture medium and agent iscapable of suppressing effector T-cell function and/or is capable ofreducing an immune response such as an immune response to anautoantigen.

In an embodiment, the composition comprises one or more of the solubleCD52 glycoprotein, fusion protein, cell culture medium or agent, and isformulated for mucosal and/or transdermal administration.

In a further embodiment, the composition further comprises insulinand/or an autoantigen.

The present disclosure also provides a fusion protein comprising solubleCD52 glycoprotein as a first protein, and a second protein.

Preferably, the soluble CD52 glycoprotein of the fusion proteincomprises an amino acid sequence which is at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% identical, or is 100% identical, to any one or more of theamino acid sequences identified in SEQ ID NOs: 3, 4, 5, 6 or 7.

Preferably, the fusion protein is capable of suppressing effector T-cellfunction and/or is capable of reducing an immune response such as animmune response to an autoantigen. In an embodiment, the fusion proteinreduces the immune response to an extent that it results in tolerance toat least one antigen such an autoantigen.

The second protein may be any protein capable of increasing thestability and/or solubility of the soluble CD52 glycoprotein, ofenhancing the process of making the soluble CD52 glycoprotein byrecombinant methods, or of enhancing the therapeutic effect of thesoluble CD52 glycoprotein. In one example, the second protein maycomprise an antibody fragment, such as an Fc.

Preferably, the fusion protein is soluble.

The present disclosure also provides an isolated or recombinantpolynucleotide encoding the fusion protein disclosed herein.

The present disclosure also provides a vector comprising thepolynucleotide disclosed herein.

The present disclosure also provides an isolated cell comprising thepolynucleotide and/or the vector disclosed herein. The cell may be amammalian cell. In one example, the cell is as HEK293T cell. In anotherexample, the cell is a Daudi B lymphoblast cell.

In addition, the present disclosure provides a method of producing thefusion protein, comprising expressing the polynucleotide or vectordisclosed herein under glycosylation-permitting conditions.

In an embodiment, the glycosylation-permitting conditions compriseexpressing the fusion protein in a host cell, such as a mammalian cell.

The present disclosure also provides for the use of any one or more of:

-   -   i) soluble CD52 glycoprotein,    -   ii) a fusion protein comprising soluble CD52 glycoprotein as a        first protein, and a second protein;    -   iii) a polynucleotide encoding the peptide portion of soluble        CD52 glycoprotein of part i) or the fusion protein of part ii);    -   iv) a vector comprising the polynucleotide of part iii);    -   v) an isolated cell comprising the polynucleotide of part iii)        or the vector of part iv);    -   vi) an isolated CD52^(hi) cell capable of producing soluble CD52        glycoprotein;    -   vii) an isolated cell population comprising a plurality of        CD52^(hi) cells capable of producing soluble CD52 glycoprotein;    -   viii) cell culture medium, or a fraction thereof comprising        soluble CD52 glycoprotein, isolated from a cell culture        comprising the cell of part vi) or the cell population of part        vii);    -   ix) an agent capable of increasing the level of expression of        soluble CD52 glycoprotein by a cell; and    -   x) the pharmaceutical composition of the invention,        to suppress effector T-cell function and/or to reduce an immune        response, such as an immune response to an autoantigen.

The present disclosure also provides a method of treating or preventinga disease or condition mediated by effector T-cell function,inflammation or sepsis, in a subject, the method comprisingadministering a therapeutically effective amount of any one or more of:

-   -   i) soluble CD52 glycoprotein,    -   ii) a fusion protein comprising soluble CD52 glycoprotein as a        first protein, and a second protein;    -   iii) a polynucleotide encoding the peptide portion of soluble        CD52 glycoprotein of part i) or the fusion protein of part ii);    -   iv) a vector comprising the polynucleotide of part iii);    -   v) an isolated cell comprising the polynucleotide of part iii)        or the vector of part iv);    -   vi) an isolated CD52^(hi) cell capable of producing soluble CD52        glycoprotein;    -   vii) an isolated cell population comprising a plurality of        CD52^(hi) cells capable of producing soluble CD52 glycoprotein;    -   viii) cell culture medium, or a fraction thereof comprising        soluble CD52 glycoprotein, isolated from a cell culture        comprising the cell of part vi) or the cell population of part        vii);    -   ix) an agent capable of increasing the level of expression of        soluble CD52 glycoprotein by a cell; and    -   x) the pharmaceutical composition of the invention,        to the subject.

In an embodiment, the soluble CD52 glycoprotein, fusion protein, cellculture medium, agent or composition is administered at a mucosal ortransdermal site.

The present disclosure also provides any one or more of:

-   -   i) soluble CD52 glycoprotein,    -   ii) a fusion protein comprising soluble CD52 glycoprotein as a        first protein, and a second protein;    -   iii) a polynucleotide encoding the peptide portion of soluble        CD52 glycoprotein of part i) or the fusion protein of part ii);    -   iv) a vector comprising the polynucleotide of part iii);    -   v) an isolated cell comprising the polynucleotide of part iii)        or the vector of part iv);    -   vi) an isolated CD52^(hi) cell capable of producing soluble CD52        glycoprotein;    -   vii) an isolated cell population comprising a plurality of        CD52^(hi) cells capable of producing soluble CD52 glycoprotein;    -   viii) cell culture medium, or a fraction thereof comprising        soluble CD52 glycoprotein, isolated from a cell culture        comprising the cell of part vi) or the cell population of part        vii);    -   ix) an agent capable of increasing the level of expression of        soluble CD52 glycoprotein by a cell; and    -   x) the pharmaceutical composition of the invention,        for use in treating or preventing a disease or condition        mediated by effector T-cell function, inflammation or sepsis.

Furthermore, the present disclosure provides for the use of any one ormore of:

-   -   i) soluble CD52 glycoprotein,    -   ii) a fusion protein comprising soluble CD52 glycoprotein as a        first protein, and a second protein;    -   iii) a polynucleotide encoding the peptide portion of soluble        CD52 glycoprotein of part i) or the fusion protein of part ii);    -   iv) a vector comprising the polynucleotide of part iii);    -   v) an isolated cell comprising the polynucleotide of part iii)        or the vector of part iv);    -   vi) an isolated CD52^(hi) cell capable of producing soluble CD52        glycoprotein;    -   vii) an isolated cell population comprising a plurality of        CD52^(hi) cells capable of producing soluble CD52 glycoprotein;    -   viii) cell culture medium, or a fraction thereof comprising        soluble CD52 glycoprotein, isolated from a cell culture        comprising the cell of part vi) or the cell population of part        vii);    -   ix) an agent capable of increasing the level of expression of        soluble CD52 glycoprotein by a cell; and    -   x) the pharmaceutical composition of the invention,        in the manufacture of a medicament for the treatment or        prevention of a disease or condition mediated by effector T-cell        function, inflammation or sepsis.

In an embodiment, the medicament is formulated for administration at amucosal or transdermal site.

In one example, the disease mediated by effector T-cell function is anautoimmune disease, such as type I diabetes or rheumatoid arthritis. Inanother example, the condition mediated by effector T-cell function isan allograft rejection or a graft-versus-host reaction.

The present disclosure also provides a method of diagnosing a subject'ssusceptibility to developing a disease or condition mediated by effectorT-cell function, inflammation or sepsis, the method comprising:

detecting the level of soluble CD52 glycoprotein in a sample taken fromthe subject; and

comparing the level of soluble CD52 glycoprotein detected in the sampletaken from the subject with a reference level determined from one ormore healthy subjects,

wherein a lower level of soluble CD52 glycoprotein detected in thesample taken from the subject compared to the reference level indicatesthat the subject has an increased susceptibility to developing a diseaseor condition mediated by effector T-cell function, inflammation orsepsis.

The present disclosure also provides a method of diagnosing a subject'ssusceptibility to developing a disease or condition mediated by effectorT-cell function, inflammation or sepsis, the method comprising:

detecting the frequency of CD52^(hi) cells in a sample taken from asubject; and

comparing the frequency of CD52^(hi) cells detected in the sample takenfrom the subject with a reference level determined from one or morehealthy subjects, wherein a lower frequency of CD52^(hi) cells detectedin the sample taken from the subject compared to the reference levelindicates that the subject has an increased susceptibility to developinga disease or condition mediated by effector T-cell function,inflammation or sepsis.

The present disclosure also provides a method of diagnosing a subject'ssusceptibility to developing a disease or condition mediated by effectorT-cell function, inflammation or sepsis, the method comprising:

detecting the activity of CD52^(hi) cells in a sample taken from asubject; and

comparing the activity of CD52^(hi) cells detected in the sample takenfrom the subject with a reference level determined from one or morehealthy subjects, wherein a reduced activity of CD52^(hi) cells detectedin the sample taken from the subject compared to the reference levelindicates that the subject has an increased susceptibility to developinga disease or condition mediated by effector T-cell function,inflammation or sepsis.

In one example, the frequency of CD52^(hi) cells is determined bydetecting the level of membrane bound CD52 in the sample, by detectingthe level of expression of CD52 protein in the sample, and/or bydetecting the level of expression of CD52 mRNA in the sample.

In an embodiment, the sample is taken from a subject to which an antigenhas been administered.

In another embodiment, the sample is taken from a local disease site inthe subject.

The present disclosure also provides a method of determining a subject'ssuitability for entry into a drug screening trial, comprising performingthe method of the invention and identifying the subject as being moresuitable for entry into a drug screening trial if the subject has alower level of soluble CD52 glycoprotein, a lower frequency of CD52^(hi)cells, or a reduced activity of CD52^(hi) cells than the referencesample. For example, the drug screening trial is an anti-diabetic drugscreening trial.

In addition, the present disclosure also provides a method ofidentifying an agent capable of mimicking the effectorT-cell-suppressing, and/or immune response suppressing, function of asoluble CD52 glycoprotein, the method comprising determining whether atest agent suppresses effector T-cell function and/or an immuneresponse.

The present disclosure also provides a method of identifying a potentialtherapeutic agent for the treatment or prevention of a disease orcondition mediated by effector T-cell function, inflammation or sepsis,the method comprising contacting a test agent with a CD52^(hi) cell orCD52^(hi) cell population, and detecting any one or more of the level ofsoluble CD52 glycoprotein produced by the cell or cell population, thefrequency of CD52^(hi) cells and/or the activity of CD52^(hi) cells, andidentifying the test agent as a potential therapeutic agent for thetreatment or prevention of a disease or condition mediated by effectorT-cell function, inflammation or sepsis, if the level of soluble CD52glycoprotein, the frequency of CD52^(hi) cells and/or the activity ofCD52^(hi) cells is increased after contact with the test agent.

The features of any embodiment described herein shall be taken to applymutatis mutandis to any other embodiment unless specifically statedotherwise.

The present disclosure is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1: GAD65-specific CD4⁺ suppressor T-cell clones display higherexpression of CD52.

(A) Proliferation of a GAD65-specific T-cell clone in the presence of anautologous suppressor clone. GAD65 used was human recombinant glutamicacid decarboxylase 65. A fixed number (25,000) of GAD65-specificnon-Treg clone (3.19) cells was co-cultured with increasing numbers ofan autologous GAD65-specific Treg clone (1.4) in the presence or absenceof GAD65 and irradiated PBMCs (100,000) as antigen presenting cells.³H-thymidine uptake was measured after 72 hr. Results (mean±sem oftriplicates) are representative of multiple autologous suppressor andnon-suppressor clone pairs as previously described (Dromey et al.,2011). (B) Activated GAD65-specific suppressor clones have higherexpression of CD52. Flow cytometric histograms of CD52 expression byautologous GAD65-specific suppressor (solid line) and non-suppressor(dashed line) clones following overnight stimulation by plate-boundanti-CD3 antibody. Staining by isotype control antibody is shown ingrey. The result is representative of 3 clone pairs from 3 individuals.

FIGS. 2A-2D: High expression of CD52 is a marker of antigen-activatedblood CD4⁺ T-cells with suppressor function.

FIG. 2A. Proliferation of tetanus toxoid (TT)-stimulated, FACS-sortedCD4⁺ T-cells re-activated with TT in the presence of GAD65-activated andsorted CD52^(hi) or CD52^(lo) CD4⁺ cells. Activated CD4⁺ cells weregenerated by incubating CFSE-labelled PBMCs with either GAD65 or TT for7 days (A1). GAD65-activated CD52^(hi) CD4⁺ and CD52^(lo) CD4⁺ T-cells,and TT-activated CD4⁺ T-cells, were then isolated by FACS. In thepresence of GAD65, proliferation of cells re-activated by TT issuppressed by GAD65-activated CD52^(hi) CD4⁺ cells. 3H-thymidine uptakewas measured over the last 16 h of a 3-day culture (A2). Results(mean±sem of triplicates) are representative of independent experimentson cells from 5 individuals.

FIG. 2B. IFN-γ-secretion by GAD65-activated and sorted CD4⁺ T-cells inthe absence or presence of GAD65. CF SE-labelled PBMCs were incubatedwith GAD65 for 7 days and sorted into CD52^(hi) and CD52^(lo) CD4⁺T-cells. Sorted cells (5,000) were incubated in ELISpot plates withirradiated PBMCs (20,000). Results (mean+sem of triplicates) arerepresentative of multiple independent experiments on cells from 5individuals.

FIG. 2C. IFN-γ secretion by TT-activated and sorted CD4⁺ T-cells in theabsence or presence of TT±IL-2 (10 U/ml). As in FIG. 2B, except thatCD52^(hi) and CD52^(lo) CD4⁺ populations were sorted from CFSE-labelledPBMCs activated by TT. Results are mean+sem of triplicates.

FIG. 2D. Proliferation of PBMCs initially depleted by FACS of eitherCD52^(hi) or CD52^(lo) CD4⁺ cells before CFSE labelling and incubationin the absence or presence of GAD65 for 7 days. Results arerepresentative of two experiments.

FIG. 3: CD4+CD52^(hi) T-cells are not derived from resting CD4^(|)CD25^(|) T-cells. IFN-γ-secretion by TT-activated and sorted CD4⁺T-cells in the absence (open bars) or presence (filled bars) of TT,after initially depleting CD25^(hi) cells from PBMCs.

FIG. 4: Antigen-activated CD52^(hi) CD4⁺ T-cells are not distinguishedby markers of conventional CD4⁺ CD25⁺ Treg cells.

Flow cytometric expression of (A) CD25, (B) FoxP3, (C) surface and (D)intracellular CTLA-4, (E) GITR, (F) CD127, (G) CD24 and (H) CD59 ondivided CD52^(hi) (black line) and CD52^(lo) (grey line) CD4⁻ T-cells,following incubation of PBMCs with TT for 7 days. Staining by isotypecontrol antibody is shown as grey fill. Results are representative of 5individuals.

FIG. 5: CD52 gene expression is higher in CD52^(hi) CD4⁺ T-cellsrelative to CD52^(lo) CD4⁺ T-cells.

Expression of genes in CD52^(hi) relative to CD52^(lo) CD4⁺ T-cells.Quantitative RT-PCR was performed in triplicate RNA samples extractedfrom sorted CFSE-labelled CD52^(hi) and CD52^(lo) CD4⁺ T-cells fromthree individuals, 7 days after activation by GAD65. Results areexpressed as median+interquartile range.

FIG. 6: CD24 expression does not delineate CD52^(hi) CD4⁺ T-cells withsuppressor function.

IFN-γ secretion by TT-activated and sorted CD52^(lo) CD4⁺ T-cellsre-stimulated with TT in the presence of TT-stimulated and sorted CD52and CD24 subpopulations. Results are mean+sem of triplicates.

FIG. 7: Cell-cell contact is not required for suppression by CD52^(hi)CD4⁺ T-cells.

FIGS. 8A-8B: Release of soluble CD52 accounts for suppression byCD52^(hi) CD4⁺ T-cells.

FIG. 8A Immunoblotting of media conditioned by GAD65-activated CD52^(hi)or CD52^(lo) CD4⁺ T-cells then re-activated by GAD65. CFSE-labelledPBMCs were incubated with GAD65 for 7 days and sorted into CD52 andCD52^(lo) CD4⁻ T-cells. Sorted cells were re-activated with GAD65 andmedia collected after 24 hrs. Media were concentrated 10-fold,fractionated by SDS-PAGE, transferred to a PDVF membrane and blottedwith a rabbit polyclonal antibody to CD52. The approximate molecularweight of native soluble CD52 is indicated.

FIG. 8B Immunoblotting of media conditioned by TT-activated PBMCs +/−the phospholipase C inhibitor U73122. CFSE-labelled PBMCs were incubatedwith TT and media collected after 1 hr was processed as in (A).

FIG. 8C Effect of phospholipase C inhibitor on suppression byTT-activated CD52^(hi) CD4⁺ T-cells. CFSE-labelled PBMCs were incubatedwith TT for 7 days and sorted into CD52^(hi) and CD52^(lo) CD4⁻ T-cells,which then were incubated together (5,000 of each) in ELISpot plateswith irradiated PBMCs (20,000) and TT±the phospholipase C inhibitorU73122. Results are mean+sem of triplicates. There was no effect ofU73122 in the absence of TT.

FIG. 8D Effect of antibody to the carbohydrate moiety of CD52 onsuppression by TT-activated CD52^(hi) CD4⁻ T-cells. Procedures were asin (C) except that cells in the ELISpot assay were incubated with orwithout TT and either 10 μg/ml anti-CD52 (CF1D12) or isotype control(IgG3) monoclonal antibody. Results (mean±sem) are representative ofthree independent experiments.

FIGS. 9A-9B: Soluble CD52 produced from Daudi cells directly suppressesT-cell proliferation and effector function.

FIG. 9A Immunoblotting of media conditioned by cells lines. Media wereconcentrated 10-fold, fractionated by SDS-PAGE, transferred to a PDVFmembrane and blotted with a rabbit polyclonal antibody to CD52.

FIG. 9B Suppression of T-cell proliferation by Daudi cell conditionedmedium. PBMCs (200,000 cells) were cultured for 7 days in IMDMcontaining 20% Daudi cell conditioned medium with TT and eitheranti-CD52 (CF1D12) or isotype control antibody (10 μg/mL). To depletesoluble CD52, Daudi medium was incubated overnight with rabbit anti-CD52polyclonal antibody (1 μg/ml medium) followed by precipitation withprotein G-Sepharose for 1 h at 4° C. Results (mean±sem) arerepresentative of three independent experiments.

FIG. 10: DNA constructs for expression in lentivirus vector.

SigP=signal peptide; ECD=extracellular domain; Strep2=purification tagencoding 8 amino acids that binds to Strep-Tactin, a specificallyengineered streptavidin.

FIGS. 11A-11D: Soluble CD52 fusion protein directly suppresses T-cellproliferation and effector function.

Suppression of T-cell proliferation by recombinant CD52-Fc. PBMCs(200,000) were cultured with TT for 7 days (FIG. 11A) and purified CD4⁺T-cells (20,000) with anti-CD3 (100 ng/ml) and anti-CD28 (200 ng/ml)antibody for 48 hrs (FIG. 11B), with 4 times the number of irradiatedPBMCs in 200 μl round bottom wells, in the presence of recombinantCD52-Fc or Fc protein control protein at the indicated concentrations.³H-thymidine uptake was measured over the final 16 hrs of incubation.Results (mean±sem of triplicates) are representative of six independentexperiments.

FIG. 11C Suppression of cytokine secretion by recombinant CD52-Fc. Mediafrom PBMCs activated with TT in (C)±3.3 μM CD52-Fc or Fc proteins weresampled after 48 hrs incubation and assayed for cytokines by multiplexbead array.

FIG. 11D Impaired suppression by CD52-Fc after cleavage of N-linkedcarbohydrate. CD52-Fc (20 μg) was incubated with or without PNGase F(1,000 units) in 20 μl PBS for 1 h at 37° C., and the reactionterminated by heating at 75° C. for 10 min. PBMCs incubated with TT andtreated or untreated CD52-Fc (final 2.5 μM) for 7 days at 37° C., and3H-thymidine uptake then measured as in (FIG. 11C). Upper panel showsthe determination by SDS-PAGE and Coomassie staining of the decrease insize of CD52-Fc after PNGase F treatment.

FIGS. 12A-12E: CD52 carbohydrate binding to Siglec-10 is required forsoluble CD52 effector function.

FIG. 12A Suppression of T-cell activation by CD52-Fc±treatment withneuraminidase. CD52-Fc (3.3 μM) was incubated with neuraminidase (1unit) or carrier buffer only in 20 μl for 30 min at 37° C. PBMCs werethen incubated with TT ±neuraminidase-treated or untreated CD52-Fc(final 0.33 μM) in a 48-well plate for 1 h at 37° C. before non-adherentcells were transferred to an ELISpot plate and developed after 24 h at37° C. for IFN-γ spots.

FIG. 12B Siglec-10 expression on human T-cells after T-cell activation.Flow cytometric histograms of Siglec-10 expression on CD4⁺ T-cells afterincubation of PBMCs with TT or soluble anti-CD3 antibody for 4 days.

FIG. 12C Suppression of T-cell function by CD52-Fc when co-incubatedwith anti-Siglec-10 antibody. PBMCs were incubated in an ELISpot platewith TT and CD52-Fc (3.4 μM) and different concentrations ofaffinity-purified goat antibody to the extracellular domain ofSiglec-10, or Fc (0.34 μM)±antibody before non-adherent cells weretransferred to an ELISpot plate for 24 h for development of IFN-γ spots.

FIG. 12D Suppression of T-cell function by CD52-Fc when co-incubatedwith soluble recombinant Siglec-10-Fc. PBMCs were incubated in a 48-wellplate with TT and CD52-Fc (3.4 μM) and different concentrations ofrecombinant Siglec-10-Fc before non-adherent cells were transferred toan ELISpot plate for 24 hrs for development of IFN-γ spots.

FIG. 12E Blockade of Siglec-10 but not other Siglecs reduces T-cellsuppression by CD52-Fc. CD4⁺ T-cells (20,000) were incubated intriplicate ELISpot plate wells at 37° C. with TT, together with CD52-Fcor Fc (3.4 μM each) and anti-human Siglec antibodies (10 μg/ml each) orrecombinant human Siglec 2-Fc (20 μg/ml), as indicated. After 20 hrs,wells were washed and developed for IFN-γ spots.

FIG. 13: CD52-Fc does not affect the T-cell stimulatory capacity ofpurified blood dendritic cells.

FACS-sorted human blood CD1b/c⁺ DC were pre-incubated with CD52-Fc or Fcprotein, washed twice and co-cultured with allogeneic CFSE-labeled CD4+T-cells for 6 days. The frequency of dividing CD4⁺ T-cells identified asCFSE^(lo) was determined by flow cytometry. The result is representativeof two independent experiments with different donors. Similar resultswere obtained for CD304⁺ plasmacytoid DC and for CD14⁺ monocytes (datanot shown).

FIG. 14: Transfer of CD52^(hi)-depleted splenocytes induces rapid onsetof diabetes in NOD.SCID mice.

Total splenocytes from wild-type NOD mice depleted or sham depleted ofCD52^(hi) cells were injected iv into (A) 8 week-old RIP.B7/NOD.SCIDmice (2×10⁶ cells) and (B) irradiated (750 rad) 8 week-old male NOD mice(1.2×10⁷ cells). Mice were monitored by measuring urine glucose twiceweekly using Diastix (Bayer) and diabetes confirmed by a blood glucosemeasurement >14 mM on consecutive days. Results show percentage survivalover time after transfer of the respective cell populations.

FIG. 15: CD52^(hi)-depleted CD3⁺ T-cells accelerate onset of diabetes inirradiated NOD mice.

Irradiated (750 rad) 8 week-old male NOD mice were injected with 1.2×10⁷splenocytes or CD3⁺ CD52^(hi) depleted splenocytes derived from 10week-old non-diabetic female NOD mice. Mice were monitored by measuringurine glucose twice weekly using Diastix (Bayer) and diabetes confirmedby a blood glucose measurement >14 mM on consecutive days. Results show(A) percentage survival over time after transfer of the respective cellpopulations and (B) insulitis score (n=4/group) after 4 weeks.

FIG. 16: The frequency of CD52^(hi) CD4⁺ T-cells generated in responseto simulation by GAD65 is impaired in type 1 diabetes.

The proportion of CD52^(hi) CD4⁺ T-cells expanded from PBMCs in responseto (A) GAD65 and (B) TT is shown for individuals in the followingcategories: Pre-T1D—at risk for type 1 diabetes; T1D—with type 1diabetes; Healthy—disease-free HLA DR3 and/or DR4 young adults; T2D—type2 diabetes. The horizontal bar is the median for each group. Overall Pvalues for analysis of variance shown were determined by theKruskal-Wallis test; Dunn's multiple comparison test then revealedsignificant differences between both Pre-T1D and T1D compared Healthy orT2D at P<0.05.

FIG. 17: Suppression by CD52^(hi) CD4⁺ cells generated in response toGAD65 is impaired in pre-clinical T1D.

IFN-γ-secretion by TT- or GAD65-activated and sorted CD4⁺ T-cells in theabsence (open) or presence (filled) of the antigen. Results (mean+sem oftriplicates) are representative of experiments on cells from sixindividuals with islet cell autoantibodies at risk for type 1 diabetes.

FIG. 18: Treatment with CD52-Fc reverses hyperglycemia in NOD mice withrecent-onset diabetes.

Blood glucose levels in female NOD mice were monitored by weekly testingfor urine glucose and diabetes was diagnosed in mice with a positiveurine test by a blood glucose concentration >14 mM. As soon ashyperglycemia was confirmed mice were given either CD52-Fc or Fc, 20 μgi.p., six doses on alternate days, and their blood glucoseconcentrations then monitored twice weekly. Results are shown for twopairs of mice that received either CD52-Fc (A) or Fc control (B).

FIG. 19: Development of diabetes in NOD.SCID mice after transfer fromdiabetic NOD mice of splenocytes treated ex vivo with hCD52-Fc or Fc.

Recombinant human CD52 Fc- or Fc-treated diabetic NOD splenocytes wereinjected into NOD.SCID mice. Splenocytes from female diabetic mice wereisolated and incubated with either recombinant hCD52-Fc or Fc protein in‘CD52 buffer’. Cells were re-suspended and injected into male NOD.SCIDmice (6 per group; see Methods Example 18).

FIG. 20: Human CD52-Fc suppresses proliferation of mouse ovalbumin(Ova)-specific TCR transgenic CD4 (OT-II) T-cells.

Splenocytes (1×10⁵) from 10 week-old female OT-II mice were incubatedfor 3 days in round bottom 96-well plates in 200 ml RPMI-1640 mediumcontaining 5% FCS and the indicated concentrations of ova protein (A) orpeptide (B), or anti-CD3 antibody (clone 2C-11) (C), and recombinanthuman CD52-Fc or Fc protein. ³H-thymidine uptake was measured over thelast 16 h of culture. Results are mean±sem of triplicates.

FIG. 21: Identification by ELISA of CD52 inhuman semen samples.Absorbance at 450 nm is shown for soluble CD52 in serial dilutions ofsemen samples (n=26).

FIG. 22: Semen-derived CD52 suppresses human T-cell proliferation.

The effect on T-cell proliferation (Cell Division Index; CDI) calculatedfrom CFSE dye dilution in response to tetanus toxoid (TT, 5 Lfu/ml) isshown for two semen samples (at a dilution of 1:20) withoutimmunodepletion or depleted with control IgG (‘Octagam’) or anti-CD52IgG (Campath).

FIG. 23: T-cell proliferation (CFSE dye dilution) to tetanus toxoid:effect of semen±blocking antibody to CD52.

The effect on T-cell proliferation (CDI, calculated from CFSE dyedilution) in response to tetanus toxoid (TT, 5 Lfu/ml) is shown forsemen samples (1:20)±blocking antibody CF1D12 (20 μg/ml).

FIG. 24: hCD52-Fc suppresses IL-1β secretion by THP 1 cells in responseto LPS. THP-1 cells were incubated with different doses of CD52-Fc or Fccontrol in presence of LPS, medium collected and the concentration ofIL-1B measured by ELISA.

FIG. 25: hCD52-Fc suppresses IL-1β secretion by THP1 cells in responseto Pam3CSK.

THP-1 cells were incubated with different doses of CD52-Fc or Fc controlin presence of the TLR-2 agonist Pam3CSK, media collected and theconcentration of IL-1β measured by ELISA.

FIG. 26: hCD52-Fc (50 μg/ml) suppresses IL-1β secretion bydifferentiated THP1 cells in response to alum.

THP-1 cells were differentiated with phorbol-12-myristate-13-acetate(PMA), washed and incubated with CD52-Fc or Fc control. Medium wascollected and the concentration of IL-1β measured by ELISA.

FIG. 27: mCD52-Fc suppresses secretion of IL-1β by mouse bonemarrow-derived dendritic cells in response to a range of innate immunestimuli.

Bone marrow-derived dendritic cells (BMDCs) from C57/B6 mice wereincubated with mouse CD52-Fc or PBS (Control) in presence of LPS, CPG orListeria monocytogenes, primed with LPS and then stimulated with theknown inflammsome agonists, monosodium urate (MSU), alum and nigericin.Cytokine concentrations in the media were measured by multiplex cytokinearray assay.

FIG. 28: Treatment with A. ureafaciens neuraminidase abolishes thesuppressive effect of mCD52-Fc on LPS-induced IL-1,8 production by THP-1cells.

THP-1 cells were incubated with neuraminidase- or reactionbuffer-treated mCD52-Fc in presence of LPS. Media were collected and theconcentration of IL-1β measured by ELISA.

FIG. 29: Treatment with PNGase-F to remove N-linked oligosaccharideabolishes the suppressive effect of hCD52-Fc on LPS-induced IL-1βsection by THP-1 cells. Human CD52-Fc (300 μg) treated with or withoutPNGase F to remove N-linked oligosaccharide was used to treat THP-1cells in the presence of LPS. Media were collected and the concentrationof IL-1β measured by ELISA.

KEY TO THE SEQUENCE LISTING

SEQ ID NO: 1 Human CD52 mRNA transcript (NCBI Reference Sequence:NM_001803.2)

SEQ ID NO: 2 Amino acid sequence of human CD52

SEQ ID NO: 3 12 amino acid soluble peptide of human CD52

SEQ ID NO: 4 Orthologous monkey soluble CD52 peptide

SEQ ID NO: 5 Orthologous mouse soluble CD52 peptide

SEQ ID NO: 6 Orthologous rat soluble CD52 peptide

SEQ ID NO: 7 Orthologous dog soluble CD52 peptide

SEQ ID NO: 8 CD52 F primer

SEQ ID NO: 9 CD52 R primer

SEQ ID NO: 10 FOXP3 F primer

SEQ ID NO: 11 FOXP3 R primer

SEQ ID NO: 12 CTLA-4 F primer

SEQ ID NO: 13 CTLA-4 R primer

SEQ ID NO: 14 GITR F primer

SEQ ID NO: 15 GITR R primer

SEQ ID NO: 16 CD127 F primer

SEQ ID NO: 17 CD127 R primer

SEQ ID NO: 18 IL-2α forward primer

SEQ ID NO: 19 IL-2α reverse primer

SEQ ID NO: 20 IL-27β forward primer

SEQ ID NO: 21 IL-27β reverse primer

SEQ ID NO: 22 IL-12α forward primer

SEQ ID NO: 23 IL-12α reverse primer

SEQ ID NO: 24 1F1 primer

SEQ ID NO: 25 1R1 primer

SEQ ID NO: 26 1F2 primer

SEQ ID NO: 27 1R2 primer

SEQ ID NO: 28 2F primer

SEQ ID NO: 29 2R1 primer

SEQ ID NO: 30 2R2 primer

SEQ ID NO: 31 CD52 forward primer

SEQ ID NO: 32 CD52 reverse primer

SEQ ID NO: 33 IL-2 forward primer

SEQ ID NO: 34 IL-2 reverse primer

SEQ ID NO: 35 IL-4 forward primer

SEQ ID NO: 36 IL-4 reverse primer

SEQ ID NO: 37 IL-10 forward primer

SEQ ID NO: 38 IL-10 reverse primer

SEQ ID NO: 39 IL-13 forward primer

SEQ ID NO: 40 IL-13 reverse primer

SEQ ID NO: 41 FoxP3 forward primer

SEQ ID NO: 42 FoxP3 reverse primer

SEQ ID NO: 43 CD127 forward primer

SEQ ID NO: 44 CD127 reverse primer

SEQ ID NO: 45 CTLA-4 forward primer

SEQ ID NO: 46 CTLA-4 reverse primer

SEQ ID NO: 47 FASLG forward primer

SEQ ID NO: 48 FASLG reverse primer

SEQ ID NO: 49 TGFb1 forward primer

SEQ ID NO: 50 TGFb1 reverse primer

SEQ ID NO: 51 TGFb2 forward primer

SEQ ID NO: 52 TGFb2 reverse primer

SEQ ID NO: 53 IFNg forward primer

SEQ ID NO: 54 IFNg reverse primer

SEQ ID NO: 55 IL-12 alpha forward primer

SEQ ID NO: 56 IL-12 alpha reverse primer

SEQ ID NO: 57 Ebi3 forward primer

SEQ ID NO: 58 Ebi3 reverse primer

SEQ ID NO: 59 RARA forward primer

SEQ ID NO: 60 RARA reverse primer

SEQ ID NO: 61 GITR forward primer

SEQ ID NO: 62 GITR reverse primer

SEQ ID NO: 63 GRANZMB forward primer

SEQ ID NO: 64 GRANZMB reverse primer

SEQ ID NO: 65 ALDH1A2 forward primer

SEQ ID NO: 66 ALDH1A2 reverse primer

SEQ ID NO: 67 ACTIN forward primer

SEQ ID NO: 68 ACTIN reverse primer

SEQ ID NO: 69 Human Siglec-10 protein sequence (GenBank Accession No.AF310233.1)

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, immunology, immunohistochemistry, protein chemistry,and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal., (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term “about”, unless stated to the contrary, refersto +/−20%, more preferably +/−10%, more preferably +/−5%, of thedesignated value. For the avoidance of doubt, the term “about” followedby a designated value is to be interpreted as also encompassing theexact designated value itself (for example, “about 10” also encompasses10 exactly).

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

As used herein, the term “immune response” has its ordinary meaning inthe art, and includes both humoral and cellular immunity. An immuneresponse may be mediated by one or more of, T-cell activation, B-cellactivation, natural killer cell activation, activation of antigenpresenting cells (e.g., B cells, DCs, monocytes and/or macrophages),cytokine production, chemokine production, specific cell surface markerexpression, in particular, expression of co-stimulatory molecules. In apreferred embodiment, the immune response which is suppressed using themethods of the invention is at least effector T cell function byreducing the survival, activity and/or proliferation of this cell type.In another preferred embodiment, the immune response which is suppressedusing the methods of the invention is at least one or more of monocyte,macrophage or dendritic cell function by reducing the survival, activityand/or proliferation of one or more of these cell types. In a furtherpreferred embodiment, the immune response is suppressed to an extentsuch that it induces tolerance to an antigen such as an autoantigen.

As used herein, “tolerance” refers to a state of immune unresponsivenessto a specific antigen or group of antigens to which a subject isnormally responsive. Immune tolerance is achieved under conditions thatsuppress the immune reaction and is not just the absence of an immuneresponse.

As used herein, the terms “treating”, “treat” or “treatment” includeadministering a therapeutically effective amount of an agent sufficientto reduce or eliminate at least one symptom of disease.

As used herein, the terms “preventing”, “prevent” or “prevention”include administering a therapeutically effective amount of an agentsufficient to prevent the manifestation of at least one symptom ofdisease.

As used herein, the term “suppressing” includes reducing by anyquantifiable amount.

As used herein, the term “subject” refers to an animal, e.g., a mammal.In a preferred embodiment, the subject is mammal, for example a human.Other preferred embodiments include livestock animals such as horses,cattle, sheep and goats, as well as companion animals such as cats anddogs.

As used herein, the term “host” refers to any organism from whichsoluble CD52 can be isolated or in which soluble CD52 can be produced,by any means. The host may be whole organism or may be a cell derivedtherefrom. The host may be an animal, e.g., a mammal. In a preferredembodiment, the host is mammalian, for example a human. Other preferredhosts include mice, rats, monkeys, hamsters, guinea-pigs, rabbits, andany animal or cell which may serve as a suitable host from which solubleCD52 can be isolated or in which soluble CD52 can be produced.

As used herein, the terms “linked”, “attached”, “conjugated”, “bound”,“coupled” or variations thereof are used broadly to refer to any form ofcovalent or non-covalent association which joins one entity to anotherfor any period of time.

Soluble CD52

The present disclosure describes, for the first time, the suppression ofimmune cells such as effector T-cells, monocytes and dendritic cells bya soluble CD52 glycoprotein fragment. CD52 is a surfaceglycosylphosphatidylinositol (GPI)-anchored glycoprotein present on mostlymphoid cells, initially recognised as the target of complement-bindingCAMPATH monoclonal antibodies used therapeutically to depletelymphocytes (Treumann et al., 1995; Xia et al., 1991; Hale, 2001). ThemRNA transcript of the human CD52 gene is shown in SEQ ID NO: 1 and thetranslated amino acid sequence is shown in SEQ ID NO: 2. Mature CD52tethered by its GPI anchor comprises only 12 amino acids and anasparagine (N)-linked terminal carbohydrate.

Unless stated otherwise, the terms “soluble CD52 glycoprotein”, “solubleCD52”, “soluble glycoprotein” and variations thereof are usedinterchangeably herein.

Membrane-anchored CD52 can be cleaved (for example, enzymatically) torelease a soluble peptide fragment comprising the amino acid sequenceGQNDTSQTSSPS (SEQ ID NO: 3). The soluble CD52 glycoprotein disclosedherein may comprise an amino acid sequence at least 60% identical to theamino acid sequence of SEQ ID NO: 3 or at least 60% identical to theamino acid sequence of other known, orthologous CD52 soluble fragmentsequences. Thus, orthologous sequences of the soluble CD52 peptidefragment are encompassed by the present disclosure. Such sequencesinclude but are not limited to the monkey sequence SQNATSQSSPS (SEQ IDNO: 4), the mouse sequence GQATTAASGTNKNSTSTKKTPLKS (SEQ ID NO: 5), therat sequence GQNSTAVTTPANKAATTAAATTKAAATTATKTTTAVRKTPGKPPKA (SEQ ID NO:6), the dog sequence GNSTTPRMTTKKVKSATPA (SEQ ID NO:7), and otherorthologous sequences readily identifiable from known CD52 polypeptideand polynucleotide sequences.

Percentage identity to any of the amino acid or polynucleotide sequencesdisclosed herein may be determined by methods known in the art. Forexample, amino acid and polynucleotide sequences can be comparedmanually or by using computer-based sequence comparison andidentification tools that employ algorithms such as BLAST (Basic LocalAlignment Search Tool; Altschul et al., 1993); see alsowww.ncbi.nlm.nih.gov/BLAST/), the Clustal method of alignment (Higginsand Sharp, 1989) and others, wherein appropriate parameters for eachspecific sequence comparison can be selected as would be understood by aperson skilled in the art. The amino acid sequence of the peptideportion of the glycoprotein disclosed herein can be at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% identical, or at least 99% identical to any one or more of theamino acid sequences identified in SEQ ID NOs: 3, 4, 5, 6 or 7. Forexample, the amino acid sequence of the peptide portion of theglycoprotein disclosed herein can be 100% identical to any one of theamino acid sequences identified in SEQ ID NOs: 3, 4, 5, 6 or 7.

Isolated soluble CD52 glycoprotein may be used to produce pharmaceuticalcompositions of the invention. The term “isolated” is used herein todefine the isolation of the soluble CD52 glycoprotein so that it ispresent in a form suitable for application in a pharmaceuticalcomposition. Thus, the glycoprotein disclosed herein is isolated fromother components of a host cell or fluid or expression system to theextent that is required for subsequent formulation of the glycoproteinas a pharmaceutical composition. The isolated glycoprotein is thereforeprovided in a form which is substantially free of other components of ahost cell (for example, proteins) which may hinder the pharmaceuticaleffect of the glycoprotein. Thus, the isolated glycoprotein may be freeor substantially free of material with which it is naturally associatedsuch as other glycoproteins, polypeptides or nucleic acids with which itis found in its natural environment, or the environment in which it isprepared (e.g. cell culture) when such preparation is by recombinant DNAtechnology practised in vitro or in vivo. Soluble glycoprotein can beisolated from a host cell or fluid or expression system by methods knownin the art.

The term “soluble” is used herein to define a peptide or glycoproteinwhich is not bound to a cell membrane. The soluble peptide orglycoprotein may be able to move freely in any solvent or fluid, such asa bodily fluid. For example, the soluble peptide or glycoprotein may beable to circulate in blood.

The carbohydrate may be any carbohydrate moiety attached to the solubleCD52 peptide fragment. For example, the carbohydrate may be anycarbohydrate moiety found to be attached to the extracellular portion ofthe CD52 protein in a host. Thus, the carbohydrate may be anycarbohydrate capable of being attached to the extracellular portion ofthe CD52 protein by a glycosylation reaction known to take place in ahost.

Carbohydrate moieties present on a naturally occurring CD52 glycoproteincan be identified by known methods, such as those described in Schroteret al. (1999). Such carbohydrate moieties may be identified from CD52glycoproteins present in any host cell expressing CD52, and particularlylymphocytes, such as CD4⁺ or CD8⁺ T-cells, monocytes or genital tractcells, such as sperm cells or epididymal duct cells. Thus, the precisestructure of the carbohydrate moiety can be determined by applyingmethods such as mass spectrometry (e.g. Matrix-assisted LaserDesorption/Ionization—Time of Flight Mass Spectrometry (MALDI-TOF)),Mono-Q anion-exchange chromatography, high pH anion exchangechromatography (HPAEC-PAD), methylation analysis, endo-β-galactosidasedigestion, and other methods. The N-glycans may be separated from anaturally occurring CD52 glycoprotein using known cleavage enzymes suchas peptide-N4-(N-acetyl-β-D-glucosaminyl)asparagines amidase F (‘PNGaseF’ from Flavobacterium meningosepticum, recombinant from Escherichiacoli; obtainable from commercial suppliers such as Roche). The N-glycanscan be isolated for further characterisation using known chromatographicmethods, such as C8-reversed phase chromatography. In one example, thecarbohydrate may comprise one or more bi-, tri- or tetra-antennarysugars, which may be terminally sialylated. For example, thecarbohydrate may comprise one or more tetra-antennary sugars. The sugarsmay be branched or unbranched. The sugars may comprise a proximalfucose. Thus, the carbohydrate may be fucosylated. The sugars maycomprise one or more N-acetyllactosamine repeats. Thus, the sugars maycomprise polylactosamine units. In addition, the sugars may comprise amannose core.

The carbohydrate may have any one or more of the structures described inTreumann et al. (1995). Thus, for example, the carbohydrate may have anyof the following structures:

Thus, the carbohydrate may comprise one or more sialic acids. The one ormore sialic acids may be located in any portion of the carbohydrate. Forexample, the one or more sialic acids may be terminal sialic acids. Inone particular example, the carbohydrate may comprise terminal α2-6sialic acids. Thus, the carbohydrate may comprise one or more surfaceα2-6-sialyllactose groups. The one or more sialic acids may be attachedto galactose in β1-4 linkage with N-acetylglucosamine.

The present disclosure demonstrates that the soluble CD52 glycoproteinexerts its suppressive effect at least in part via binding to the sialicacid binding Ig-like lectin-10 (Siglec-10), a cell surface transmembranereceptor and immunoglobulin superfamily member bearing two cytoplasmicimmunoreceptor tyrosine-based inhibition motifs (ITIMs) (Munday et al.,2001; Crocker et al., 2007). Thus, the soluble glycoprotein disclosedherein may be capable of binding to Siglec-10. For example, the solubleglycoprotein disclosed herein may comprise a carbohydrate moiety capableof binding to Siglec-10. In one example, the carbohydrate moietycomprises one or more surface α2-6- or α2-3-sialyllactose groups thatare capable of binding to Siglec-10. Alternatively, the carbohydratemoiety may comprise any other surface groups that are capable of bindingto Siglec-10.

The soluble glycoprotein disclosed herein may be capable of binding toSiglec-10 derived from any species. For example, the solubleglycoprotein disclosed herein may be capable of binding to Siglec-10derived from any mammalian species. Preferably, the soluble glycoproteindisclosed herein is capable of binding to human Siglec-10. Thepolypeptide sequence of human Siglec-10 is defined in Munday et al.(2001), in GenBank Accession No. AF310233.1, and in SEQ ID NO: 69.

The soluble glycoprotein disclosed herein may be capable of effectingsignalling via the Siglec-10 receptor. Thus, the soluble glycoproteindisclosed herein may be capable of binding to Siglec-10 to any extentsufficient to effect signalling via the Siglec-10 receptor. Thus, theprecise level of binding to Siglec-10 can vary. Methods for determiningwhether a given glycoprotein is capable of binding to Siglec-10, and fordetermining whether a given glycoprotein is capable of effectingsignalling via the Siglec-10 receptor are known in the art.

Further examples of the N-linked CD52 carbohydrate which theglycoprotein disclosed herein may comprise are those derived orderivable from host lymphocyte CD52 glycoproteins or genital tract cellCD52 glycoproteins.

Due to the complex nature of many naturally occurring carbohydratemoieties known to be linked to the extracellular protein portion ofhuman CD52 and the many variations in these structures that may arisefrom varying glycosylation patterns, it will be understood that theprecise nature of the carbohydrate moiety present in the glycoproteindisclosed herein may vary. As stated above, methods are available toprecisely identify particular carbohydrate moieties from naturallyoccurring CD52 glycoproteins. In addition, a number of differentcarbohydrate moieties can be added to the soluble peptide fragment ofCD52 by expressing CD52 under varying glycosylation conditions. Forexample, the soluble glycoprotein disclosed herein may be expressed inand/or isolated from host lymphocyte cells, monocytes or host genitaltract cells (e.g. sperm cells, or epididymal duct cells) or seminalfluid and may therefore comprise different carbohydrate groups as aresult. The inventors have shown that soluble CD52 present in humansemen, similarly to soluble CD52 released from lymphocytes such as DaudiB cells, is capable of suppressing T-cell function and/or an immuneresponse. Alternative host cells providing different glycosylationconditions may be selected for expression of soluble CD52 in order toprovide alternative forms of carbohydrate on the soluble glycoprotein.

The carbohydrate may be attached to any one or more amino acid in thepeptide which is capable of having a carbohydrate moiety attachedthereto. For example, the carbohydrate may be attached to one or moreasparagine, serine, threonine, tyrosine, hydroxylysine, hydroxyproline,phosphoserine or tryptophan residues if present in the amino acidsequence. In one example, the carbohydrate is attached to the asparagine(N) residue in a peptide portion having a sequence at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% identical, or is 100% identical, to the aminoacid sequence set out in SEQ ID NO: 3.

The present disclosure also provides variants, mutants, biologicallyactive fragments, modifications, analogs and/or derivatives of theglycoprotein disclosed herein. Such compounds can be identified byscreening for compounds which mimic the structure and/or function of thepolypeptide disclosed herein, using methods including any of the methodsdisclosed herein.

Soluble CD52 Function

The glycoprotein disclosed herein is preferably capable of suppressingthe activity (“function”) of immune cells including lymphocytes (such asa T-cell) and monocytes. For example, the glycoprotein disclosed hereinis capable of suppressing one or more of effector T-cell, monocyte,macrophage and dendritic cell function. Effector T-cells, monocytes,macrophages and dendritic cells and their functions will be known to aperson skilled in the art.

T-cells can be readily identified by the presence of any of one or moreT-cell markers known in the art. The glycoprotein disclosed herein iscapable of reducing T-cell proliferation in response to antigenchallenge, and/or capable of reducing T-cell cytokine production (suchas production of any one or more of IFN-γ, IL-2, IL-10, IL-17, G-CSF,TNF-α, and other cytokines known to be secreted by activated T-cells).For example, soluble CD52 is capable of reducing IFN-γ production byT-cells.

In another example, soluble CD52 is capable of reducing IL-1β secretionby monocytes, macrophages and dendritic cells.

Accordingly, the glycoprotein disclosed herein is capable of reducing animmune response in a host. The inventors have shown that theglycoprotein disclosed herein is capable of reducing effector T-cellfunction in response to challenge with any antigen. The suppressivefunction is not dependent on the particular antigen used in thechallenge. Thus, the glycoprotein disclosed herein is capable ofreducing an immune response to any antigen. In one example, the antigenis an autoantigen.

Any known methods of determining the suppression of T-cell functionand/or an immune response can be used, such as (but not limited to)those described in the examples herein. Thus, the methods may comprisedetermining the effect of the glycoprotein disclosed herein on one ormore of effector T-cell, monocyte, macrophage and dendritic cellproliferation and/or on the production of any one or more of IFN-γ,IL-2, IL-10, IL-17, G-CSF, TNF-α, and other cytokines known to besecreted by activated T-cells, monocytes, macrophages or dendriticcells.

Fusion Proteins

The peptide portion of the CD52 glycoprotein disclosed herein may, forexample, be conjugated to a second protein as a fusion protein. Thesecond protein may be any protein capable of increasing the stabilityand/or solubility of the glycoprotein, of enhancing the process ofmaking the glycoprotein by recombinant methods, or of enhancing thetherapeutic effect of the glycoprotein. Thus, the second protein maycapable of increasing the half life of the glycoprotein disclosedherein.

The second protein can be of any suitable length. In one embodiment, thesecond protein may be relatively short. For example, the second proteinmay consist of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more aminoacids. The second protein may comprise at least 1, at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9 or at least 10 amino acids. The second protein may also comprise morethan 10 amino acids. For example, the second protein may comprise atleast 10, at least 15, at least 20, at least 25, at least 30, or atleast 50 amino acids.

In one example, the second protein is an antibody fragment. Suitableantibody fragments include any antibody fragment that is capable ofactivating the immune system. The antibody fragment may be a fragmentcrystallizable (Fc) region (which can be a single polypeptide) or anyone or more heavy chain constant domains (e.g. C_(H) domains 2, 3 and/or4) from an Fc region. In one example, the second protein is an Fcfragment.

In another example, the second protein may be a purification tag. Manyexamples of purification tags are known, and include (withoutlimitation) a His tag, T7 tag, FLAG tag, S-tag, HA tag, c-Myc tag, DHFR,a chitin binding domain, a calmodulin binding domain, a cellulosebinding domain, a Strep 2 tag (a purification tag encoding eight aminoacids that binds to Strep-Tactin, a specifically engineered streptavidin(Schmidt and Skerra, 2007), and others.

The second protein may increase the solubility of the expressed protein.Such proteins include (without limitation) NusA, thioredoxin, smallubiquitin-like modifier (SUMO), ubiquitin and others known in the art.

The second protein may increase the solubility of the expressed proteinas well as enhancing purification methods. Such proteins include(without limitation) GST, MBP, T7 gene 10, and others known in the art.

The purification tag may optionally be removed from the fusion proteinafter its production. Suitable methods of removing a purification tagfrom a fusion protein will vary depending on the particular purificationtag used. Such methods will be generally known in the art.

The fusion protein disclosed herein may comprise one or more of any ofthe second proteins described above, in any combination. Thus, thefusion protein may comprise an antibody fragment (such as an Fc) and apurification tag (such as a Strep 2 tag).

Polynucleotides

The present disclosure further provides isolated or recombinantpolynucleotides encoding the protein component of the soluble CD52glycoprotein, or the fusion protein. The sequences of suchpolynucleotides are derivable from the amino acid sequences of the CD52protein and soluble CD52 peptide fragment described herein and of thesecond protein comprised within the fusion protein. The polynucleotidesdisclosed herein may also encode a full length CD52 protein, which may,for example, be a mature form thereof, or a precursor thereof.

The term “isolated polynucleotide” is intended to mean a polynucleotidewhich has generally been separated from the polynucleotide sequenceswith which it is associated or linked in its native state. Preferably,the isolated polynucleotide is at least 60% free, more preferably atleast 75% free, and more preferably at least 90% free from othercomponents with which it is naturally associated. Furthermore, the term“polynucleotide” is used interchangeably herein with the terms “nucleicacid molecule”, “gene” and “mRNA”.

The term “recombinant” in the context of a polynucleotide refers to thepolynucleotide when present in a cell, or in a cell-free expressionsystem, in an altered amount compared to its native state. In oneembodiment, the cell is a cell that does not naturally comprise thepolynucleotide. However, the cell may be a cell which comprises anon-endogenous polynucleotide resulting in an altered, preferablyincreased, amount of production of the encoded polypeptide. Arecombinant polynucleotide of the invention includes polynucleotideswhich have not been separated from other components of the transgenic(recombinant) cell, or cell-free expression system, in which it ispresent, and polynucleotides produced in such cells or cell-free systemswhich are subsequently purified away from at least some othercomponents.

“Polynucleotide” refers to an oligonucleotide, a polynucleotide or anyfragment thereof. It may be DNA or RNA of genomic or synthetic origin,double-stranded or single-stranded, and combined with carbohydrate,lipids, protein, or other materials to perform a particular activitydefined herein.

The polynucleotides disclosed herein may possess, when compared tonaturally occurring molecules (such as genomic polynucleotides encodingCD52 or a soluble fragment thereof), one or more mutations which aredeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, made by performingsite-directed mutagenesis or DNA shuffling techniques as broadlydescribed by Harayama (1998)). It is thus apparent that polynucleotidesof the invention can be either naturally occurring or recombinant.

The particular sequence of the polynucleotide can be determined from thepeptide sequence. Due to the redundancy of the genetic code, differentsequences can be used to encode the same peptide. In addition, thepolynucleotide sequence may be specifically altered so as to enhance itsexpression in a particular host cell. Such a process is well known inthe art as “codon optimization”. Thus, the polynucleotide disclosedherein may be codon optimized to enhance expression in a host cell.

Vectors

The polynucleotide disclosed herein can be inserted into a nucleotidevector in order to facilitate expression of the protein component of theglycoprotein or the fusion protein. Accordingly, the present disclosureprovides a vector comprising a polynucleotide encoding the proteincomponent of the glycoprotein disclosed herein or the fusion proteindisclosed herein. The vector can be either RNA or DNA, eitherprokaryotic or eukaryotic, and may be a transposon (such as described inU.S. Pat. No. 5,792,294), a virus or a plasmid.

Preferably, the polynucleotide encoding the protein component of theglycoprotein or the fusion protein is operably linked to a promoterwhich is capable of expressing the peptide under suitable conditions.“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of a transcriptional regulatoryelement to a transcribed sequence. For example, a promoter is operablylinked to a coding sequence, such as a polynucleotide defined herein, ifit stimulates or modulates the transcription of the coding sequence inan appropriate cell or cell-free expression system. Generally, promotertranscriptional regulatory elements that are operably linked to atranscribed sequence are physically contiguous to the transcribedsequence, i.e., they are cis-acting. However, some transcriptionalregulatory elements, such as enhancers, need not be physicallycontiguous or located in close proximity to the coding sequences whosetranscription they enhance.

The vector is preferably an expression vector. As used herein, anexpression vector is a DNA or RNA vector that is capable of transforminga host cell and of effecting expression of a specified polynucleotidemolecule. Preferably, the expression vector is also capable ofreplicating within the host cell. Expression vectors can be eitherprokaryotic or eukaryotic, and are typically viruses or plasmids. Theexpression vectors disclosed herein include any vectors that function(i.e., direct gene expression) in the recombinant cells disclosed herein(including in animal cells) or in a suitable cell-free expressionsystem.

In particular, the expression vectors disclosed herein may containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell orcell-free expression system and that control the expression ofpolynucleotide molecules disclosed herein. In particular, the vectorsdisclosed herein may include transcription control sequences.Transcription control sequences are sequences which control theinitiation, elongation, and termination of transcription. Particularlyimportant transcription control sequences are those which controltranscription initiation, such as promoter, enhancer, operator andrepressor sequences. Suitable transcription control sequences includeany transcription control sequence that can function in at least one ofthe recombinant cells or cell-free expression systems described herein.A variety of such transcription control sequences are known to thoseskilled in the art.

The vectors disclosed herein may also contain (a) secretory signals(i.e., signal segment nucleic acid sequences) to enable an expressedprotein or peptide to be secreted from a cell that produces the peptideand/or (b) fusion sequences which lead to the expression of peptidesdisclosed herein as fusion proteins. Examples of suitable signalsegments include any signal segment capable of directing the secretionof a glycoprotein or fusion protein disclosed herein. The vectors mayalso include intervening and/or untranslated sequences surroundingand/or within the nucleic acid sequence(s) encoding the peptidedisclosed herein.

The polynucleotide or vector can be expressed in a host cell or in acell-free expression system in order to produce the glycoprotein orfusion protein disclosed herein. Such expression may be performed, forexample in a mammalian cell, a baculovirus expression system, a fungalexpression system (which may be selected so as to permit glycosylationof the expressed protein).

The host cell can be any cell capable of producing the glycoprotein orfusion protein disclosed herein. Thus, in one example, the host cell iscapable of permitting glycosylation of the protein component of theglycoprotein disclosed herein. Suitable host cells can be readilyidentified by the skilled artisan, and include, for example, animalcells, such as mammalian cells. In one example, the host cell is a CHOcell, a myeloma cell (such as the mouse myeloma NS—O or SP2-O cells) ora HEK293T cell. In another example, the host cell is a Daudi Blymphoblast cell (Hu et al., 2009).

In addition, the polynucleotide or vector can be introduced into a hostcell for administration to a subject. Thus, the pharmaceuticalcomposition disclosed herein may comprise a cell comprising thepolynucleotide or vector disclosed herein. The cell may be an isolatedcell. The cell is preferably a recombinant cell. Thus, the cell ispreferably transfected with a polynucleotide or vector disclosed herein.Any host cell suitable for administration to a subject may be used. Inone example, the cell may be a cell taken from the subject to betreated. Thus, the cell may be an autologous cell. Accordingly, one ormore cells may be taken from a subject, a polynucleotide or vector asdisclosed herein may be introduced into the subject's cell, and the cellmay then be administered to the same subject. In one example, the cellmay be a lymphocyte, such as a T-cell, such as a CD4⁺ T-cell. Methodsfor taking a suitable cell sample from a subject in this regard will beknown in the art. Where the cell to be used is a lymphocyte, the methodsmay include lymphocytapheresis. Other suitable host cells, which neednot necessarily be derived from the subject to be treated, can equallybe used. Expression of the polynucleotide or vector in the cellpreferably results in the production and/or secretion of theglycoprotein disclosed herein.

Transformation of a polynucleotide into a host cell can be accomplishedby any suitable method known in the art. Transformation techniquesinclude, but are not limited to, transfection, electroporation,microinjection, lipofection, and adsorption. A recombinant cell mayremain unicellular or may grow into a tissue, organ or a multicellularorganism. Transformed polynucleotide molecules as disclosed herein canremain extrachromosomal or can integrate into one or more sites within achromosome of the transformed (i.e., recombinant) cell in such a mannerthat their ability to be expressed is retained.

Suitable host cells to transform include any cell that can betransformed with a polynucleotide as disclosed herein. Host cells can beeither endogenously (i.e., naturally) capable of producing polypeptidesof the present invention or can be rendered capable of producing suchpolypeptides after being transformed with at least one polynucleotidemolecule as disclosed herein.

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide molecule by manipulating, for example, thenumber of copies of the polynucleotide molecule within a host cell, theefficiency with which those polynucleotide molecules are transcribed,the efficiency with which the resultant transcripts are translated, andthe efficiency of post-translational modifications. Recombinanttechniques useful for increasing the expression of polynucleotidemolecules as disclosed herein include, but are not limited to,operatively linking polynucleotide molecules to high-copy numberplasmids, integration of the polynucleotide molecule into one or morehost cell chromosomes, addition of vector stability sequences toplasmids, substitutions or modifications of transcription controlsignals (e.g., promoters, operators, enhancers), substitutions ormodifications of translational control signals (e.g., ribosome bindingsites, Shine-Dalgarno sequences), modification of polynucleotidemolecules as disclosed herein to correspond to the codon usage of thehost cell, and the deletion of sequences that destabilize transcripts.

The host cell may be cultured under conditions effective to produce theglycoprotein or fusion protein. Once expressed in the host cell, theglycoprotein or fusion protein can be isolated by conventional methodsknown in the art. Thus, in one embodiment, an isolated glycoprotein orfusion protein as described herein is produced by culturing a cellcapable of expressing the glycoprotein or fusion protein underconditions effective to produce the glycoprotein or fusion protein, andisolating the glycoprotein or fusion protein. Effective cultureconditions include, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit glycoprotein or fusionprotein production, and in particular, that permit glycosylation. Aneffective medium refers to any medium in which a cell is cultured toproduce a glycoprotein or fusion protein as disclosed herein. Suchmedium typically comprises an aqueous medium having assimilable carbon,nitrogen and phosphate sources, and appropriate salts, minerals, metalsand other nutrients, such as vitamins. Host cells can be cultured inconventional fermentation bioreactors, shake flasks, test tubes,microtiter dishes, and Petri plates. Culturing can be carried out at atemperature, pH and oxygen content appropriate for a recombinant cell.Such culturing conditions are within the expertise of one of ordinaryskill in the art.

Any cell-free expression system suitable for the expression of thepolynucleotide disclosed herein can also be used. Suitable cell-freeexpression systems include those that permit glycosylation of theprotein component of the glycoprotein or fusion protein. Such conditionscan be determined by a person skilled in the art.

The glycoprotein disclosed herein may also be produced by inducingexpression of CD52 in an isolated host cell and isolating soluble CD52glycoprotein produced by the host cell. Thus, the glycoprotein may beproduced using a cell which naturally produces soluble CD52. Suitablecells will be identifiable to the person skilled in the art and include(without limitation) lymphocytes, cells of the genital tract area (suchas sperm cells), Daudi B lymphoblast cells (Hu et al., 2009), K562cells, and others. Additional cell lines capable of naturally producingsoluble CD52 can be identified by screening for soluble CD52 secretion.Thus, cancer cells can be screened for their ability to secrete solubleCD52.

The methods of producing the glycoprotein disclosed herein from anisolated host cell which naturally produces soluble CD52 may comprisestimulating the host cell to produce higher levels of soluble CD52. Thismay be achieved, for example, by contacting the host cell with anantigen. Any antigen may be used. In one example, the antigen is anautoantigen, such as GAD65. In another example, the antigen is tetanustoxoid.

The methods of producing the glycoprotein disclosed herein from anisolated host cell which naturally produces soluble CD52 may alsocomprise selecting a cell which naturally expresses CD52 and contactingthe cell with an enzyme capable of cleaving the extracellular portion ofmembrane-bound CD52 to release soluble CD52. Suitable enzymes are knownin the art and include phospholipases such as phospholipase C.

The methods described herein can be performed on isolated cells or cellpopulations of a size sufficient to produce the desired quantity ofsoluble CD52.

CD52^(hi) Cells

The present disclosure also provides isolated cells and cell populationsexhibiting high levels of expression of CD52. By “high” it is meant thatthe expression levels of CD52 are relatively high compared to CD52expression levels in a given population of cells. The given populationof cells may be, for example, a population of lymphocytes. Thelymphocyte population may comprise Treg cells and non-Treg cells. Inaddition, the lymphocyte population may have been contacted with anantigen in order to stimulate lymphocyte activity. Alternatively, thepopulation of cells may be cells of the genital tract, such as spermcells. By contrast, CD52^(lo) cells exhibit relatively low levels ofCD52 relative to a given population of cells.

In one example, a cell may be determined to be a CD52^(hi) cell if thelevel of expression of CD52 in that cell falls within the top 1%, 5%,10%, 20%, 30%, 40% or 50% CD52 expression levels in a population ofcells. Preferably, a CD52^(hi) cell has an expression level within thetop 10% of CD52 expression observed in a population of cells.

In one example, a cell may be determined to be a CD52^(lo) cell if thelevel of expression of CD52 in that cell falls within the bottom 1%, 5%,10%, 20%, 30%, 40% or 50% CD52 expression levels in a population ofcells. Preferably, a CD52^(lo) cell has an expression level within thebottom 10% of CD52 expression observed in a population of cells.

The CD52^(hi) cell may be isolated from the population of cells fromwhich it is identified. Alternatively, a population of CD52^(hi) cellsmay be isolated from the initial cell population from which theCD52^(hi) cells are identified. Thus, the cell populations disclosedherein may be enriched for CD52^(hi) cells.

The present disclosure therefore provides an isolated cell populationcomprising a plurality of CD52^(hi) cells. The CD52^(hi) cells maycomprise at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% ofthe total enriched cell population.

The isolated CD52^(hi) cells and populations of CD52^(hi) cellsdisclosed herein are capable of producing the soluble CD52 glycoproteindisclosed herein.

These isolated cells and cell populations may be further defined by thepresence of one or more additional cell markers. In one example, theCD52^(hi) cells are CD4⁺ CD52^(hi) cells. Alternatively, the CD52^(hi)cells are CD8⁺ CD52^(hi) cells. Additional markers that characterisethese cells include any one or more of glucocorticoid-induced tumornecrosis factor receptor related protein (GITR), CD127, Fas ligand (FasLor CD95L), sphingosine-1-phosphate receptor (S1PR), the GPI-anchoredglycoprotein CD24, CD25, FoxP3, CTLA-4, and other markers, in anycombination. The inventors have found that GITR, CD127, Fas L, S1PR andCD24 expression levels may be higher in CD52^(hi) Treg cells compared toCD52^(lo) cells. These markers can therefore be used to further define aCD52^(hi) cell or a CD52^(hi) cell population as described herein.

In addition, the function of a given cell may be used to define aCD52^(hi) cell or a CD52^(hi) cell population as described herein. Forexample, the ability of a cell expressing CD52 to reduce effector T-cellfunction as described herein can be used to identify a CD52^(hi) cell ora CD52^(hi) cell population.

Cell Culture Medium

CD52^(hi) cells or a CD52^(hi) cell population as described herein maybe cultured so as to produce medium comprising the soluble glycoproteindisclosed herein. Suitable culture conditions will be apparent to theperson skilled in the art. The cultured cells may additionally beinduced to increase their level of expression of soluble CD52 by anysuitable method, including by contacting the cells with antigen.

Ex Vivo Cell Treatment

The present invention also provides a pharmaceutical compositioncomprising cells, preferably immune cells, and a pharmaceuticallyacceptable carrier, wherein the cells have been treated ex vivo with anyone or more of:

-   -   i) soluble CD52 glycoprotein,    -   ii) a fusion protein comprising soluble CD52 glycoprotein as a        first protein, and a second protein;    -   iii) a polynucleotide encoding the peptide portion of soluble        CD52 glycoprotein of part i) or the fusion protein of part ii);    -   iv) a vector comprising the polynucleotide of part iii);    -   v) an isolated cell comprising the polynucleotide of part iii)        or the vector of part iv);    -   vi) an isolated CD52^(hi) cell capable of producing soluble CD52        glycoprotein;    -   vii) an isolated cell population comprising a plurality of        CD52^(hi) cells capable of producing soluble CD52 glycoprotein;    -   viii) cell culture medium, or a fraction thereof comprising        soluble CD52 glycoprotein, isolated from a cell culture        comprising the cell of part vi) or the cell population of part        vii); and    -   ix) an agent capable of increasing the level of expression of        soluble CD52 glycoprotein by a cell;

The cells of the composition may be, for example, whole blood or acellular fraction thereof such as peripheral blood mononuclear cells(PBMCs).

Such ex vivo treated cells can be used in the present invention, forexample for treating or preventing a disease or condition mediated byeffector T-cell function, inflammation or sepsis.

In one embodiment, the cells are autologous in respect to the subject towhich they will be administered. In another embodiment, the cells areallogeneic.

Pharmaceutical Compositions

The present disclosure provides a pharmaceutical composition comprisingany one or more of the soluble CD52 glycoprotein, fusion protein,polynucleotide, vector, cell, cell populations and cell medium describedherein, and any agent capable of increasing the level of expression ofCD52 in a cell, and a pharmaceutically acceptable carrier.

A pharmaceutically acceptable carrier includes a carrier suitable foruse in administration to animals, such as mammals and at leastpreferably humans. In one example, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like.

Therapeutic compositions can be prepared by mixing the desired compoundshaving the appropriate degree of purity with optional pharmaceuticallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, 16^(th) edition, Osol, A. ed. (1980)), in theform of lyophilized formulations, aqueous solutions or aqueoussuspensions. Acceptable carriers, excipients, or stabilizers arepreferably nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as Tris, HEPES, PIPES, phosphate,citrate, and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyl dimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Additional examples of such carriers includeion exchangers, alumina, aluminum stearate, lecithin, serum proteins,such as human serum albumin, buffer substances such as glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, and cellulose-based substances.

A pharmaceutical composition as disclosed herein is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral (e.g., intravenous, intradermal,subcutaneous, intramuscular, intraperitoneal, intrathecal), mucosal(e.g., oral, rectal, intranasal, buccal, vaginal, respiratory), enteral(e.g., orally, such as by tablets, capsules or drops, rectally) andtransdermal (topical, e.g., epicutaneous, inhalational, intranasal,eyedrops, vaginal). Solutions or suspensions used for parenteral,intradermal, enteral or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Transdermal delivery is accomplished by exposing the therapeutic agentto a patient's skin for an extended period of time. Transdermal patcheshave the added advantage of providing controlled delivery of apharmaceutical agent to the body (see, for example, Transdermal andTopical Drug Delivery: From Theory to Clinical Practice, Williams (ed),Pharmaceutical Press, UK (2003); Transdermal Drug Delivery:Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.),Marcel Dekker, Inc., (1989)). For example, a simple adhesive patch canbe prepared from a backing material and an acrylate adhesive. Thetherapeutic agent and any enhancer are formulated into the adhesivecasting solution and allowed to mix thoroughly. The solution is castdirectly onto the backing material and the casting solvent is evaporatedin an oven, leaving an adhesive film. The release liner can be attachedto complete the system.

Alternatively, a polyurethane matrix patch can be employed to deliverthe therapeutic agent. The layers of this patch comprise a backing, apolyurethane drug/enhancer matrix, a membrane, an adhesive, and arelease liner. The polyurethane matrix is prepared using a roomtemperature curing polyurethane prepolymer. Addition of water, alcohol,and complex to the prepolymer results in the formation of a tacky firmelastomer that can be directly cast only the backing material.

A further embodiment of this invention will utilize a hydrogel matrixpatch. Typically, the hydrogel matrix will comprise alcohol, water,drug, and several hydrophilic polymers. This hydrogel matrix can beincorporated into a transdermal patch between the backing and theadhesive layer.

For passive delivery systems, the rate of release is typicallycontrolled by a membrane placed between the reservoir and the skin, bydiffusion from a monolithic device, or by the skin itself serving as arate-controlling barrier in the delivery system (see U.S. Pat. Nos.4,816,258; 4,927,408; 4,904,475; 4,588,580, 4,788,062). The rate ofdelivery will be dependent, in part, upon the nature of the membrane.For example, the rate of delivery across membranes within the body isgenerally higher than across dermal barriers.

Suitable permeable membrane materials may be selected based on thedesired degree of permeability, the nature of the complex, and themechanical considerations related to constructing the device. Exemplarypermeable membrane materials include a wide variety of natural andsynthetic polymers, such as polydimethylsiloxanes (silicone rubbers),ethylenevinylacetate copolymer (EVA), polyurethanes,polyurethane-polyether copolymers, polyethylenes, polyamides,polyvinylchlorides (PVC), polypropylenes, polycarbonates,polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulosetriacetate and cellulose nitrate/acetate, and hydrogels, e.g.,2-hydroxyethylmethacryl ate (HEMA).

Other items may be contained in the device, such as other conventionalcomponents of therapeutic products, depending upon the desired devicecharacteristics. For example, the compositions according to theinvention may also include one or more preservatives or bacteriostaticagents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate,chlorocresol, benzalkonium chlorides, and the like. These pharmaceuticalcompositions also can contain other active ingredients such asantimicrobial agents, particularly antibiotics, anesthetics, analgesics,and antipruritic agents.

Another embodiment of this invention provides for the topical deliveryof pharmaceutical composition. This treatment regimen is suitable eitherfor the systemic administration of the pharmaceutical agent or forlocalized therapy, i.e., directly to pathological or diseased tissue.Topical preparations can be prepared by combining the pharmaceuticalagent-chemical modifier complex with conventional pharmaceuticaldiluents and carriers commonly used in topical dry, liquid, cream andaerosol formulations. Ointment and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Such bases may include water and/or anoil such as liquid paraffin or a vegetable oil such as peanut oil orcastor oil. Thickening agents which may be used according to the natureof the base include soft paraffin, aluminum stearate, cetostearylalcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenatedlanolin, beeswax, and the like. Lotions may be formulated with anaqueous or oily base and will, in general, also include one or more ofthe following: stabilizing agents, emulsifying agents, dispersingagents, suspending agents, thickening agents, coloring agents, perfumes,and the like. Powders may be formed with the aid of any suitable powderbase, e.g., talc, lactose, starch, and the like. Drops may be formulatedwith an aqueous base or non-aqueous base also comprising one or moredispersing agents, suspending agents, solubilizing agents, and the like.

Dosage forms for the topical administration include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required. The ointments, pastes,creams and gels also may contain excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, talc and zincoxide, or mixtures thereof. Powders and sprays also can containexcipients such as lactose, talc, aluminum hydroxide, calcium silicatesand polyamide powder, or mixtures of these substances. Sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Mucosal (for example, gastrointestinal, sublingual, buccal, nasal,pulmonary, vaginal, corneal, and ocular membranes) drug deliveryprovides for an efficient entry of active substances to systemiccirculation and reduce immediate metabolism by the liver and intestinalwall flora (see, for example, Lee, 2001; Song et al., 2004; Hearnden etal., 2012) Transmucosal drug dosage forms (e.g., tablet, suppository,ointment, gel, salves, creams, pessary, membrane, and powder) aretypically held in contact with the mucosal membrane and disintegrateand/or dissolve rapidly to allow immediate systemic absorption.

For delivery to the buccal or sublingual membranes, typically an oralformulation, such as a lozenge, tablet, or capsule will be used. Themethod of manufacture of these formulations are known in the art,including but not limited to, the addition of the harmaceuticalagent-chemical modifier complex to a pre-manufactured tablet; coldcompression of an inert filler, a binder, and either a pharmaceuticalagent-chemical modifier complex or a substance containing the complex(as described in U.S. Pat. No. 4,806,356) and encapsulation.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound is incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions are also prepared usinga fluid carrier for use as a mouthwash, wherein the compound in thefluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Another oral formulation is one that can be applied with an adhesive,such as the cellulose derivative, hydroxypropyl cellulose, to the oralmucosa, for example as described in U.S. Pat. No. 4,940,587. This buccaladhesive formulation, when applied to the buccal mucosa, allows forcontrolled release of the pharmaceutical agent-chemical modifier complexinto the mouth and through the buccal mucosa.

For delivery to the nasal and/or pulmonary membranes, typically anaerosol formulation will be employed. The term “aerosol” includes anygas-borne suspended phase of the pharmaceutical agent-chemical modifiercomplex which is capable of being inhaled into the bronchioles or nasalpassages. Specifically, aerosol includes a gas-borne suspension ofdroplets of the compounds of the instant invention, as may be producedin a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosolalso includes a dry powder composition of the pharmaceuticalagent-chemical modifier complex suspended in air or other carrier gas,which may be delivered by inhalation from an inhaler device.

For mucosal or transdermal administration, penetrants appropriate to thebarrier to be permeated can be used in the formulation. Such penetrantsare generally known in the art, and include, for example, for mucosaladministration, detergents, bile salts, and fusidic acid derivatives.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, Cremophor™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier is a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity is maintained, for example, by the use of a coating suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. Prevention of theaction of microorganisms may be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid and the like. In many cases, it is preferable to includeisotonic agents, for example, sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions is brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

A pharmaceutically acceptable vehicle is understood to designate acompound or a combination of compounds entering into a pharmaceuticalcomposition which does not cause side effects and which makes itpossible, for example, to facilitate the administration of the activecompound, to increase its life and/or its efficacy in the body, toincrease its solubility in solution or alternatively to enhance itspreservation. These pharmaceutically acceptable vehicles are well knownand will be adapted by persons skilled in the art according to thenature and the mode of administration of the active compound chosen.

Pharmaceutical compositions to be used for in vivo administration shouldbe sterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The composition may be stored in lyophilized form or insolution if administered systemically. If in lyophilized form, it istypically formulated in combination with other ingredients forreconstitution with an appropriate diluent at the time for use. Anexample of a liquid formulation is a sterile, clear, colourlessunpreserved solution filled in a single-dose vial for subcutaneousinjection.

Pharmaceutical compositions generally are placed into a container havinga sterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle. Thecompositions are preferably administered parenterally, for example, asintravenous injections or infusions or administered into a body cavity.

The pharmaceutical compositions disclosed herein may further comprise anadditional therapeutic agent known to suppress effector T-cell functionand/or an immune response.

In one embodiment, the composition further comprises insulin.

In another embodiment, the composition further comprises an autoantigen.Examples of autoantigens useful in compositions of the inventioninclude, but are not limited to, those listed in Table 1 (Lernmark,2001).

TABLE 1 Recombinant or purified autoantigens recognized byautoantibodies associated with human autoimmune disorders. AutoantigenAutoimmune disease A. Cell or organ-specific autoimmunity Acetylcholinereceptor Myasthenia gravis Actin Chronic active hepatitis, primarybiliary cirrhosis Adenine nucleotide Dilated cardiomyopathy,translocator (ANT) myocarditis β-Adrenoreceptor Dilated cardiomyopathyAromatic L-amino acid Autoimmune polyendocrine decarboxylase syndrometype I (APS-I) Asialoglycoprotein receptor Autoimmune hepatitisBactericidal/permeability- Cystic fibrosis vasculitides increasingprotein (Bpi) Calcium-sensing receptor Acquired hypoparathyroidismCholesterol side-chain cleavage APS-I enzyme (CYPIIa) Collagen type IVα₃-chain Goodpasture syndrome Cytochrome P450 2D6 Autoimmune hepatitis(CYP2D6) Desmin Desmin Crohn disease, coronary artery disease Desmoglein1 Pemphigus foliaceus Desmoglein 3 Pemphigus vulgaris F-actin Autoimmunehepatitis GM gangliosides Guillain-Barré syndrome Glutamatedecarboxylase Type 1 diabetes, stiff man (GAD65) syndrome Glutamatereceptor (GLUR) Rasmussen encephalitis H/K ATPase Autoimmune gastritis17-α-Hydroxylase (CYP17) APS-I 21-Hydroxylase (CYP21) Addison diseaseIA-2 (ICA512) Type 1 diabetes Insulin Type 1 diabetes, insulinhypoglycemic syndrome (Hirata disease) Insulin receptor Type B insulinresistance, acanthosis, systemic lupus erythematosus (SLE) Intrinsicfactor type 1 Pernicious anemia Leukocyte function-associatedTreatment-resistant Lyme arthritis antigen (LFA-1) Myelin-associatedglycoprotein Polyneuropathy (MAG) Myelin basic protein Multiplesclerosis, demyelinating diseases Myelin oligodendrocyte Multiplesclerosis glycoprotein (MOG) Myosin Rheumatic fever p-80-Coilin Atopicdermatitis Pyruvate dehydrogenase Primary biliary cirrhosis complex-E2(PDC-E2) Sodium iodide symporter (NIS) Graves disease, autoimmunehypothyroidism SOX-10 Vitiligo Thyroid and eye muscle shared Thyroidassociated protein ophthalmopathy Thyroglobulin Autoimmune thyroiditisThyroid peroxidase Autoimmune Hashimoto thyroiditis Thyrotropin receptorGraves disease Tissue transglutaminase Coeliac disease Transcriptioncoactivator p75 Atopic dermatitis Tryptophan hydroxylase APS-ITyrosinase Vitiligo, metastatic melanoma Tyrosine hydroxylase APS-I B.Systemic autoimmunity ACTH ACTH deficiency Aminoacyl-tRNA histidylMyositis, dermatomyositis synthetase Aminoacyl-tRNA synthetasePolymyositis, dermatomyositis (several) Cardiolipin SLE Carbonicanhydrase II SLE, Sjögren syndrome, systemic sclerosis Collagen(multiple types) Rheumatoid arthritis (RA), SLE, progressive systemicsclerosis Centromere-associated proteins Systemic sclerosisDNA-dependent nucleosome- Dermatomyositis stimulated ATPase FibrillarinScleroderma Fibronectin SLE, RA, morphea Glucose-6-phosphate RAisomerase β32-Glycoprotein I (β32-GPI) Primary antiphospholipid syndromeGolgin (95, 97, 160, 180) Sjögren Syndrome, SLE, RA Heat shock proteinVarious immune-related disorders Hemidesmosomal protein 180 Bullouspemphigoid, herpes gestationis, cicatricial pemphigoid

Methods of Treatment

The soluble CD52 glycoprotein, fusion protein, polynucleotide, vector,cell, cell populations, cell medium and pharmaceutical compositiondescribed herein, and any agent capable of increasing the level ofexpression of CD52 in a cell, may be used to suppress effector T-cellfunction, inflammation or sepsis. Thus, the soluble CD52 glycoprotein,fusion protein, polynucleotide, vector, cell, cell populations, cellmedium and pharmaceutical composition described herein, and any agentcapable of increasing the level of expression of CD52 in a cell, may beused to treat any disease or condition mediated by effector T-cells,involving inflammation or sepsis.

In one example, the disease or condition mediated by effector T-cellsmay be an autoimmune disease, allograft rejection, a graft-versus-hostreaction, or an allergic disease. The term “autoimmune disease” refersto any disease in which the body produces an immunogenic (i.e., immunesystem) response to some constituent of its own tissue. Autoimmunediseases can be classified into those in which predominantly one organis affected (eg, hemolytic anemia and anti-immune thyroiditis), andthose in which the autoimmune disease process is diffused through manytissues (eg, systemic lupus erythematosus). The autoimmune disease maybe (but is not limited to) any one or more of insulin-dependent diabetesmellitus (or type 1 diabetes), insulin autoimmune syndrome, rheumatoidarthritis, psoriatic arthritis, chronic lyme arthritis, lupus, multiplesclerosis, inflammatory bowel disease including Crohn's disease,ulcerative colitis, celiac disease, autoimmune thyroid disease,autoimmune myocarditis, autoimmune hepatitis, pemphigus, anti-tubularbasement membrane disease (kidney), familial dilated cardiomyopathy,Goodpasture's syndrome, Sjogren's syndrome, myasthenia gravis,polyendocrine failure, vitiligo, peripheral neuropathy, autoimmnunepolyglandular syndrome type I, acute glomerulonephritis, adult-onsetidiopathic hypoparathyroidism (AOIH), alopecia totalis, Hashimoto'sthyroiditis, Graves' disease, Addison's disease, chronic berylliumsyndrome, ankylosing spondylitis, juvenile dermatomyositis,polychondritis, scleroderma, regional enteritis, distal ileitis,granulomatous enteritis, regional ileitis, and terminal ileitis,amyotrophic lateral sclerosis, ankylosing spondylitis, autoimmuneaplastic anemia, autoimmune hemolytic anemia, Behcet's disease, Celiacdisease, chronic active hepatitis, CREST syndrome, dermatomyositis,dilated cardiomyopathy, eosinophilia-myalgia syndrome, epidermolisisbullosa acquisita (EBA), giant cell arteritis, Goodpasture's syndrome,Guillain-Barr syndrome, hemochromatosis, Henoch-Schonlein purpura,idiopathic IgA nephropathy, insulin autoimmune syndrome, juvenilerheumatoid arthritis, Lambert-Eaton syndrome, linear IgA dermatosis,myocarditis, narcolepsy, necrotizing vasculitis, neonatal lupus syndrome(NLE), nephrotic syndrome, pemphigoid, pemphigus, polymyositis, primarysclerosing cholangitis, psoriasis, rapidly-progressiveglomerulonephritis (RPGN), Reiter's syndrome, stiff-man syndrome,inflammatory bowel disease, osteoarthritis, thyroiditis, and others. Inone example, the autoimmune disease is type 1 diabetes. In anotherexample, the autoimmune disease is rheumatoid arthritis. In anotherexample, the condition is an allograft rejection or a graft-versus-hostreaction. Thus, the methods disclosed herein may comprise administeringany one or more of the soluble CD 52 glycoprotein, fusion protein,polynucleotide, vector, cell, cell populations, cell medium, agent andpharmaceutical composition to a transplant recipient.

The allergic disease may be (but is not limited to) any one or more of afood allergy, airborne allergy, house dust mite allergy, cat allergy, orbee venom allergy, or other allergy.

Inflammation may arise as a response to an injury or abnormalstimulation caused by a physical, chemical, or biologic agent. Aninflammation reaction may include the local reactions and resultingmorphologic changes, destruction or removal of injurious material suchas an infective organism, and responses that lead to repair and healing.

Inflammation occurs in inflammatory disorders. The term “inflammatory”when used in reference to a disorder refers to a pathological processwhich is caused by, resulting from, or resulting in inflammation that isinappropriate or which does not resolve in the normal manner.Inflammatory disorders may be systemic or localized to particulartissues or organs. Inflammation is known to occur in many disorders(some of which are autoimmune diseases) which include, but are notlimited to, Systemic Inflammatory Response (SIRS); Alzheimer's Disease(and associated conditions and symptoms including: chronicneuroinflammation, glial activation; increased microglia; neuriticplaque formation; Amyotrophic Lateral Sclerosis (ALS), arthritis (andassociated conditions and symptoms including, but not limited to: acutejoint inflammation, antigen-induced arthritis, arthritis associated withchronic lymphocytic thyroiditis, collagen-induced arthritis, juvenilearthritis, rheumatoid arthritis, osteoarthritis, prognosis andstreptococcus-induced arthritis, spondyloarthropathies, and goutyarthritis), asthma (and associated conditions and symptoms, including:bronchial asthma; chronic obstructive airway disease, chronicobstructive pulmonary disease, juvenile asthma and occupational asthma);cardiovascular diseases (and associated conditions and symptoms,including atherosclerosis, autoimmune myocarditis, chronic cardiachypoxia, congestive heart failure, coronary artery disease,cardiomyopathy and cardiac cell dysfunction, including: aortic smoothmuscle cell activation, cardiac cell apoptosis and immunomodulation ofcardiac cell function); diabetes (and associated conditions, includingautoimmune diabetes, insulin-dependent (Type 1) diabetes, diabeticperiodontitis, diabetic retinopathy, and diabetic nephropathy);gastrointestinal inflammations (and related conditions and symptoms,including celiac disease, associated osteopenia, chronic colitis,Crohn's disease, inflammatory bowel disease and ulcerative colitis);gastric ulcers; hepatic inflammations such as viral and other types ofhepatitis, cholesterol gallstones and hepatic fibrosis; HIV infection(and associated conditions, including—degenerative responses,neurodegenerative responses, and HIV associated Hodgkin's Disease);Kawasaki's Syndrome (and associated diseases and conditions, includingmucocutaneous lymph node syndrome, cervical lymphadenopathy, coronaryartery lesions, edema, fever, increased leukocytes, mild anemia, skinpeeling, rash, conjunctiva redness, thrombocytosis); nephropathies (andassociated diseases and conditions, including diabetic nephropathy,endstage renal disease, acute and chronic glomerulonephritis, acute andchronic interstitial nephritis, lupus nephritis, Goodpasture's syndrome,hemodialysis survival and renal ischemic reperfusion injury);neurodegenerative diseases or neuropathological conditions (andassociated diseases and conditions, including acute neurodegeneration,induction of IL-I in aging and neurodegenerative disease, IL-I inducedplasticity of hypothalamic neurons and chronic stresshyperresponsiveness, myelopathy); ophthalmopathies (and associateddiseases and conditions, including diabetic retinopathy, Graves'ophthalmopathy, inflammation associated with corneal injury or infectionincluding corneal ulceration, and uveitis), osteoporosis (and associateddiseases and conditions, including alveolar, femoral, radial, vertebralor wrist bone loss or fracture incidence, postmenopausal bone loss,fracture incidence or rate of bone loss); otitis media (adult orpaediatric); pancreatitis or pancreatic acinitis; periodontal disease(and associated diseases and conditions, including adult, early onsetand diabetic); pulmonary diseases, including chronic lung disease,chronic sinusitis, hyaline membrane disease, hypoxia and pulmonarydisease in SIDS; restenosis of coronary or other vascular grafts;rheumatism including rheumatoid arthritis, rheumatic Aschoff bodies,rheumatic diseases and rheumatic myocarditis; thyroiditis includingchronic lymphocytic thyroiditis; urinary tract infections includingchronic prostatitis, chronic pelvic pain syndrome and urolithiasis;immunological disorders, including autoimmune diseases, such as alopeciaaerata, autoimmune myocarditis, Graves' disease, Graves ophthalmopathy,lichen sclerosis, multiple sclerosis, psoriasis, systemic lupuserythematosus, systemic sclerosis, thyroid diseases (e.g. goitre andstruma lymphomatosa (Hashimoto's thyroiditis, lymphadenoid goitre); lunginjury (acute hemorrhagic lung injury, Goodpasture's syndrome, acuteischemic reperfusion), myocardial dysfunction, caused by occupationaland environmental pollutants (e.g. susceptibility to toxic oil syndromesilicosis), radiation trauma, and efficiency of wound healing responses(e.g. burn or thermal wounds, chronic wounds, surgical wounds and spinalcord injuries), septicaemia, acute phase response (e.g. febrileresponse), general inflammatory response, acute respiratory distressresponse, acute systemic inflammatory response, wound healing, adhesion,immuno-inflammatory response, neuroendocrine response, fever developmentand resistance, acute-phase response, stress response, diseasesusceptibility, repetitive motion stress, tennis elbow, and painmanagement and response.

The methods of treatment may comprise administering a therapeuticallyeffective amount of any one or more of the soluble CD52 glycoprotein,fusion protein, polynucleotide, vector, cell, cell populations, cellmedium or pharmaceutical composition described herein, or any agentcapable of increasing the level of expression of CD52 in a cell, to asubject in need thereof.

The ‘therapeutically effective amount’ may be determined by a clinicianand may vary from one patient to another, depending on factors such asage, weight, gender, and other factors.

Diagnostic Methods

Based on the inventors' finding that soluble CD52 is a mediator of Tregfunction, the present disclosure also provides methods of determining asubject's susceptibility to any disease or condition mediated byeffector T-cells, inflammation or sepsis as described herein. Thediagnostic methods may be based on the detection of any one or more ofthe level of soluble CD52, the frequency of CD52^(hi) cells and thefunction of CD52^(hi) cells in a sample taken from the subject.

The level of soluble CD52 may be determined by any suitable method knownin the art. For example, the level of soluble CD52 may be determined byimmunoassay, using antibodies that bind to soluble CD52. Suitableantibodies include the humanized rat monoclonal antibody CAMPATH-1G,fluorescent-labelled mouse monoclonal antibodies to human CD52 (such asCF1D12), rabbit polyclonal antibody to CD52 (Santa Cruz Biotechnology,Santa Cruz, Calif., USA) and others.

The frequency of CD52^(hi) cells may be detected, for example, bydetecting the level of cell membrane bound CD52 in the sample, bydetecting the level of expression of CD52 protein in the sample, and/orby detecting the level of expression of CD52 mRNA in the sample.

The function of CD52^(hi) cells may be determined using any suitablemethod, including any of the methods disclosed herein.

The diagnostic methods may be performed on any suitable sample takenfrom the subject. In one example, the sample is taken from a mammaliansubject such as a human subject, and may originate from a number ofsources, including for example, peripheral blood mononuclear cells(PMBC), leukopheresis or apheresis blood product, bone marrow, cordblood, liver, thymus, tissue biopsy, tumour, lymph node tissue, gutassociated lymphoid tissue, mucosa associated lymph node tissue, spleentissue, or any other lymphoid tissue, or from any disease site,including the pancreas. In a preferred embodiment, the cell sampleoriginates from PBMC from a blood sample obtained from the peripheralblood of a subject.

The diagnostic methods may comprise detecting the level of any one ormore of soluble CD52, the frequency of CD52^(hi) cells and the functionof CD52^(hi) cells in a sample comprising PMBCs which have beencontacted with an antigen. Thus, the methods may comprise a step ofcontacting the sample with an antigen. In one example, the antigen maybe an autoantigen.

In one particular application of the diagnostic methods disclosedherein, the level of soluble CD52, the frequency of CD52^(hi) cellsand/or the function of CD52^(hi) cells may be determined in order toidentify a subject's suitability for entry into a drug screening trial.Thus, if a subject exhibits a lower level of soluble CD52 glycoprotein,a lower frequency of CD52^(hi) cells, and/or a decreased function ofCD52^(hi) cells, that subject may be identified as particularly suitablefor inclusion in a screening trial for a drug intended to be used in thetreatment of any disease or condition mediated by effector T-cells,inflammation, or sepsis, as described herein. In one example, the screenmay be performed in order to identify putative anti-diabetic drugs (inparticular, anti-type 1 diabetes drugs).

The diagnostic methods described herein may further comprise a step ofdetermining a reference level of soluble CD52, of the frequency ofCD52^(hi) cells and/or of the function of CD52^(hi) cells from a sampletaken from one or more healthy subjects. Alternatively, the referencelevel may be predetermined. Comparing the level of soluble CD52,frequency of CD52^(hi) cells and/or function of CD52^(hi) cells in asample taken from a subject to the reference level can indicate thesubject's susceptibility to any disease or condition mediated byeffector T-cells, inflammation, or sepsis, as described herein. Forexample, if the level of soluble CD52, frequency of CD52^(hi) cellsand/or function of CD52^(hi) cells in the sample taken from a subject islower than the reference level, that subject may be deemed to be moresusceptible to developing a disease or condition mediated by effectorT-cells, inflammation, or sepsis, as described herein. A greaterdifference between the sample level and the reference level may indicatea greater susceptibility of the subject to developing a disease orcondition mediated by effector T-cells, inflammation, or sepsis, asdescribed herein. It will be appreciated that the exact valuesindicating an increased risk of a subject developing a disease orcondition mediated by effector T-cells, inflammation, or sepsis, willvary depending on a number of factors including the particular diseaseor condition being diagnosed, the sample used for the diagnosis, thepopulation of healthy individuals used to prepare the reference level,and other factors as will be understood by a person skilled in the art.

The present disclosure also provides a method of screening for an agentcapable of suppressing effector T-cell function and/or an immuneresponse, the method comprising contacting a cell or cell populationdescribed herein (for example, a CD52^(hi) cell or cell population) witha test agent and subsequently detecting the level of soluble CD52, thefrequency of CD52^(hi) cells and/or the function of CD52^(hi) cells,wherein a higher level of soluble CD52 glycoprotein, a higher frequencyof CD52^(hi) cells, and/or an enhanced function of CD52^(hi) cells aftercontact with the test agent indicates that the test agent may bepotentially suitable for use as an agent capable of suppressing effectorT-cell function and/or an immune response.

In another embodiment, the present disclosure also provides a method ofidentifying an agent capable of mimicking the effectorT-cell-suppressing, and/or immune system suppressing, function of asoluble CD52 glycoprotein, the method comprising contacting a cell orcell population described herein (for example, a CD52^(hi) cell or cellpopulation) with a test agent and subsequently detecting the level ofsoluble CD52, the frequency of CD52^(hi) cells and/or the function ofCD52^(hi) cells, wherein a lower level of soluble CD52 glycoprotein, alower frequency of CD52^(hi) cells, and/or a reduced function ofCD52^(hi) cells after contact with the test agent indicates that thetest agent is capable of mimicking the effector T-cell-suppressing,and/or immune system suppressing, function of a soluble CD52glycoprotein.

The invention will now be further described with reference to thefollowing, non-limiting examples.

EXAMPLES Experimental Procedures Blood Donors

Venous blood drawn into sodium heparin tubes was obtained with informedconsent and Human Research Ethics Committee approval from 5 healthyyoung adults (3 males, 2 females) and a young adult male at risk forT1D, all known to have blood T-cell responses to GAD65. All donors hadbeen vaccinated to tetanus toxoid. Peripheral blood mononuclear cells(PBMCs) were isolated on Ficoll/Hypaque (Amersham Pharmacia Biotech AB,Uppsala, Sweden), washed twice in human tonicity phosphate bufferedsaline (PBS) and resuspended in Iscove's modified Dulbecco's medium(Gibco, Melbourne, Australia) containing 5% pooled, heat-inactivatedhuman serum, 100 mM non-essential amino acids, 2 mM glutamine and5×10⁻⁵M 2-mercaptoethanol (complete Iscove's modified Dulbecco's medium[IMDM]).

Antibodies and Other Reagents

Reagents and suppliers were as follows: fluorescent-labelled mousemonoclonal antibodies to human CD52 (clone CF1D12) and CD24 (clone SN3)(Caltag), FoxP3, GITR, ICOS, CD25,CD127 and human Siglec-10 (clone 5G6)(Biolegend, San Diego, Calif., USA); mouse IgG3 (Caltag); rabbitpolyclonal antibody to CD52 (Santa Cruz Biotechnology, Santa Cruz,Calif., USA); HRP-conjugated horse anti-rabbit IgG and anti-mouse IgG(Cell Signaling, Arundal, QLD, Australia); ECL reagent (GE Healthcare,Rydalmere, NSW, Australia), humanized rat monoclonal antibody(CAMPATH-1G) to CD52 (Bayer Healthcare, Pymble, NSW, Australia), mousemonoclonal antibodies to human IFN-γ (Mabtech, Sydney, NSW, Australia),and IL-10Ra (clone 37607), goat anti-human TGF-βRII and goataffinity-purified antibody to human Siglec-10 and recombinant humanSiglec-10-Fc (R & D Systems, Minneapolis, Minn.); IL-2 (NCIBRBPreclinical Repository, Rockville, Md.); synthetic human CD52 peptide(GL Biochem, Shanghai); indomethacin, nitro-L-arginine methylester,1-methyl-dl-tryptophan, SCH58261 (adenosine A2A receptor antagonist)(Sigma-Aldrich, St. Louis, Mo., USA); carboxyfluorescein diacetatesuccinimidyl ester (CFSE) (Molecular Probes, Eugene, Oreg., USA);neuraminidase (C. perfringens type V) (Sigma-Aldrich, Castle Hill,Australia); ³H-thymidine (ICN, Sydney, Australia); 0.4 μm Corning Costartranswells (Crown Scientific, Minto, NSW, Australia); Protein G andA-Sepharose (WEHI Monoclonal Lab, Bundoora, Victoria Australia),phospholipase C (U7322) and D (1,10-phenanthroline) inhibitors(Sigma-Aldrich Pty. Ltd. NSW, Australia), phospholipase C (MolecularProbes, Eugene, Oreg., USA), PNGase F (New England Biolabs, Ipswich,Mass., USA), Strep-Tactin Sepharose (IBA GmbH Gottingen, Germany).Tetanus toxoid (TT) was generously provided by CSL (Parkville, Victoria,Australia). Recombinant GAD65 produced in Baculovirus and purified asdescribed (Bach et al., 1997) was purchased from Dr Peter Van Endert,Hopital Necker, Paris. The endotoxin concentration of the GAD65 stocksolution, measured by Limulus lysate assay (BioWhittaker, Walkerville,Md., USA), was 1.2 EU/mg/ml. TT and GAD65 were used at concentrations of10 Lyons flocculating units (LFU)/ml and 5 μg/ml, respectively, unlessotherwise stated. Cytokines and soluble IL-2 receptor-α (CD25) wereassayed in media by Milliplex MAP bead arrays (Abacus ALS, Brisbane,Australia).

Statistical Analysis

Replicates were expressed as mean±sem. Significance between groups wasdetermined by unpaired (2-tail) Student t test, using GraphPad Prismversion 3.0cx for Macintosh (GraphPad Software Inc., San Diego, Calif.).

Example 1 Analysis of GAD65-Specific CD4⁺ T-Cell Clones Methods

GAD65-specific CD4⁺ T-cell clones, previously generated and screened forGAD65-specific suppressor function, were thawed and cultured asdescribed (Dromey et al., 2011). Initially, suppressor andnon-suppressor clones were screened for surface markers against an arrayof solid phase antibodies (Medsaic Ptd Ltd, Sydney, Australia) (Belov etal., 2003). Clones (1×10⁶) were taken directly from culture and analysedresting or after stimulation for 24 hrs with plate-bound anti-CD3 (5μg/ml). For phenotyping by flow cytometry, cells were stained on icewith the appropriate concentrations of labelled antibodies. Staining forintracellular FoxP3 and intracellular CTLA-4 was combined.

Results

Screening pairs of autologous suppressor and non-suppressor clones fordifferences in surface phenotype using a CD antibody array revealed thatactivated suppressor clones were consistently found to have higherexpression of CD52, a result which was confirmed by flow cytometry (seeFIG. 1). Thus, CD52 was identified as a potential marker of Treg cells.

Example 2 Analysis of Blood CD4⁺ CD52^(hi) T-Cells Methods

PBMCs stained with carboxyfluorescein succinimidyl ester (CFSE) werecultured in IMDM in 96-well round-bottom plates, without or with GAD65or TT, at 2×10⁵ in 200 μl in replicates of six. After 7 days, replicateswere pooled, washed in 0.1% BSA-PBS and stained on ice with anti-humanCD4-PE, -PECy7 or -APC and CD52-PE (clone CF1D12) antibodies. Viable(propidium-iodide negative) CFSE^(dim) CD4⁺ cells that had undergonedivision in response to GAD65 were sorted in a FACSAria (BD Biosciences,North Ryde, NSW, Australia) into fractions with the highest to thelowest CD52 expression, and single cells cloned as described (Dromey etal., 2011). Subsequently, in response to GAD65 or TT, CD52^(hi) andCD52^(lo) populations corresponding, respectively, to the upper 10% andlower 10% of CD52 expression on undivided CD4⁺ cells were sorted forfurther study. These cut-offs were chosen because the majority ofGAD65-specific suppressor clones generated were from the upper 10% ofCD52⁺ cells (see Table 1).

Using PBMCs from the same donor over 4 consecutive weeks, theinter-assay coefficient of variation of the CD52^(hi) to CD52^(lo) ratioin response to GAD65 was 21.8%. Resting PBMCs were sorted intoCD4⁺CD52^(hi) and CD52^(lo) cells, and also collected unsorted as acontrol. In separate experiments, prior to CFSE labelling, PBMCs weredepleted of CD25⁺ cells by AutoMACS selection (Miltenyi Biotec);isotype-matched monoclonal antibodies were used for control‘depletions’.

The function of GAD65- or TT-activated CD52^(hi) and CD52^(lo) CD4⁻cells was analysed in two ways. First, sorted CD52^(hi) or CD52^(lo)cells were co-cultured with TT-activated CD4⁺ T-cells at a 1:1 ratio(1×10⁴/well) in 6 wells of a 96-well plate. Each well also contained5×10⁴ irradiated autologous PBMCs as APCs and TT to stimulateproliferation of the autologous TT-activated CD4⁺ T-cells. GAD65 wasadded to 3 of the 6 wells to re-stimulate sorted cells. As a control,irradiated PBMCs were also cultured with or without GAD65. After 48 hrs,³H thymidine (37 kBq) was added to each well, and the cells harvested 16hrs later. Second, sorted CD52^(hi) or CD52^(lo) CD4⁺ cells (5-20,000each) were cultured alone or in combination at a 1:1 ratio in 6replicate wells of a 96-well ELISpot plate (Millipore PVDF MultiScreenHTS) containing pre-bound anti-IFN-γ antibody. Each well also containedfour times the number of irradiated autologous PBMCs as APCs. GAD65 orTT was added to 3 of the 6 wells to re-stimulate sorted cells. After 24hrs, cells were removed by washing and spots developed by incubationwith biotinylated second antibody, followed by streptavidin-alkalinephosphatase and BCIP/NBT colour reagent. Results were expressed as IFN-γspots/5,000 CD4⁺ cells.

Results

A majority (22/29, 76%) of GAD65-specific suppressor clones was found tobe derived from GAD65-activated CD4⁺ T-cells with the highest CD52expression (upper 10%) (Table 1). Thus, suppressor clones appeared to bederived from primary blood CD52^(hi)CD4⁺ T-cells rather than being anartefact of the cloning conditions.

TABLE 1 Suppressor clones derived from GAD65-activated CD4⁺ T-cellsfractionated according to CD52 expression* CD52 fraction^(†) Number ofclones (%) generated GAD65-specific suppressor clones Upper 5% 86  9(10.5) Upper 10% 94 13 (13.8) Upper 20% 87 5 (5.7) Lower 80% 60 2 (3.3)*PBMCs from a healthy individual known to have GAD65-reactive T-cellswere labeled with CFSE and incubated with GAD65 for 7 days. From eachCD52⁺ fraction, 240 single, viable (propidium-iodide negative)CFSE^(dim) CD4⁺ cells that had undergone division were FACS sorted intowells of 96-well plates and cloned as pervious described (Dromey et al,2001). ^(†)Corresponding to CD52 expression on undivided CD4⁺ T-cells.

As the majority of GAD65-specific CD4⁺ suppressor clones were derivedfrom divided cells with CD52 expression corresponding to the upper 10%on undivided CD4⁺ cells this threshold could be used to define aCD52^(hi) CD4⁺ population after activation. When re-activated withGAD65, sorted CD52 but not CD52^(lo) CD4⁺ cells suppressed proliferationof autologous TT-specific CD4⁺ T-cells (FIG. 2A). To ensure thatsuppression was specific for CD52^(hi) cells and not due to the methodof their selection GAD65-activated CD4⁻ cells were sorted for highexpression of two other GPI-anchored glycoproteins, CD24 and CD59, aswell as for CD62L, HLA-DR, CD80 and ICOS, but these populations did notsuppress proliferation of TT-specific T-cells (data not shown).

Functional differences between sorted CD52^(hi) and CD52^(lo) CD4⁺T-cells after reactivation with GAD65 were also demonstrated by ELISpotassay. A lower proportion of CD52^(hi) than CD52^(lo) cells secretedIFN-γ and addition of CD52^(hi) to CD52^(lo) cells reduced the number ofIFN-γ secreting cells in response to re-activation [compareCD52^(hi)+CD52^(lo) (p<0.002) with CD52^(lo)+CD52^(lo) cells (p<0.0002)in FIG. 2B]. Suppression was not unique to CD52^(hi) CD4⁺ T-cellsactivated by GAD65 and was also observed when tetanus toxoid (TT) wasused as the activating antigen (FIG. 2C). Because T-cell responses to TTwere stronger, subsequent studies mostly employed TT as antigen.Supplementation with a low concentration of IL-2 (10 U/ml) increased thenumber of both CD52^(hi) and CD52^(lo) IFN-γ secreting cells in responseto reactivation, but did not alter suppression by CD52^(hi) cells (FIG.2C). CD52^(hi) CD4⁺ cells that were sorted from non-activated,polyspecific PBMCs exhibited weak, usually significant suppression ofT-cells activated by GAD65 or TT (data not shown). However, afterantigen activation, suppressor CD52^(hi) CD4⁺ cells were most likelyderived from pre-existing CD52^(hi) CD4⁺ cells because depletion ofthese cells from resting PBMCs increased the response of residualT-cells to GAD65 (FIG. 2D).

Example 3 CD52^(hi) CD4⁺ T-Cells are Distinct from CD4⁺CD25⁺ Treg CellsMethods

PBMCs were labelled with anti-CD25α antibody and depleted of CD25^(hi)cells on an AutoMACS column (84% compared to isotype control antibody‘depletion’). Cells were then labelled with CFSE and incubated with TTfor 7 days before being sorted into CD52^(hi) and CD52^(lo) cells,reactivated by TT and analysed by ELISpot assay.

Results

Following depletion of CD25^(hi) cells, the proportion of dividedCD52^(hi) CD4⁺ cells in response to TT increased (18.1% versus 11.8%with control depletion) but their suppressor function after reactivationwith TT remained unchanged (FIG. 3). Thus, suppressor CD52^(hi) CD4⁺cells do not appear to be derived from the population of CD4⁺CD25⁺T-cells.

Example 4 Phenotypic Analysis of CD52^(hi) CD4⁺ T-Cells Methods

Flow cytometric expression of (A) CD25α, (B) FoxP3, (C) surface and (D)intracellular CTLA-4, (E) GITR, (F) CD127, (G) CD24 and (H) CD59 ondivided CD52^(hi) (black line) and CD52^(lo) (grey line) CD4⁺ T-cells,following incubation of PBMCs with TT for 7 days. Staining by isotypecontrol antibody is shown as grey fill. Results are representative of 5individuals.

Results

CD4⁻CD25⁺ Treg cells have high expression of CD25, FoxP3, CTLA-4 andglucocorticoid-induced tumor necrosis factor receptor related protein(GITR) (Sakaguchi et al., 2008; Shevach, 2006) and low expression ofCD127 (Seddiki et al., 2006; Liu et al., 2006). In contrast, except forhigher expression of GITR, CD52^(hi) CD4⁺ T-cells had similar expressionof CD25, FoxP3 and CTLA-4, and consistently higher expression of CD127,compared to CD52^(lo) CD4⁺ T-cells (FIG. 4). Expression of theGPI-anchored glycoprotein, CD24, structurally related to CD52 (Tone etal., 1999), was higher on CD52^(hi) CD4⁺ T-cells but this was not thecase for GPI-anchored CD59 (FIG. 4) or CD73, or for CD103, CD40, β7integrin, ICOS and PD-1 (data not shown). Thus, CD52^(hi) CD4⁺ T-cellsare a novel population of suppressor cells which are not characterizedby expression of markers used to define human CD4⁺CD25⁺ Treg cells, andwhich are detected in the context of activation by antigen, implyingthat they contribute to T-cell homeostasis during T-cell division.

Example 5 Gene Expression Analysis of CD52^(hi) CD4⁺ T-Cells Methods

The expression of the CD52 gene and of genes for proteins found to haveincreased expression on CD52^(hi) CD4⁺ T-cells was investigated byquantitative real time RT-PCR. CFSE-labelled CD52^(hi) and CD52^(lo)CD4⁺ T-cells were sorted from three individuals, 7 days after activationby GAD65. Total RNA was extracted from cells with the RNAeasy Mini Kit(Qiagen, Melbourne, Australia), treated with RNase-free DNase (Qiagen)and quantified with the Agilent 2100 Bioanalyser. cDNA was reversetranscribed from 10 ng RNA/reaction. Primers for PCR, designed withPrimerExpress software and synthesized by Sigma-Aldrich (Castle Hill,NSW, Australia), were:

(SEQ ID NO: 8) CD52 F: CAA ACT GGA CTC TCA GGA CAA A  (SEQ ID NO: 9)CD52 R: CAA CTG AAG CAG AAG AGG TGG A  (SEQ ID NO: 10)FOXP3 F: ATG GTT TCT GAA GAA GGC AAA C  (SEQ ID NO: 11)FOXP3 R: GGA CTA CTT CAA GTT CCA CAA CA  (SEQ ID NO: 12)CTLA-4 F: AAC CTA CAT GAT GGG GAA TGA G  (SEQ ID NO: 13)CTLA-4 R: TTA CAT AAA TCT GGG TTC CGT T  (SEQ ID NO: 14)GITR F: GGG AAA TTC AGT TTT GGC TTC  (SEQ ID NO: 15)GITR R: ACA GCG TTG TGG GTC TTG TT  (SEQ ID NO: 16)CD127 F: CCT TTT GAC CTG AGT GTC GTC T  (SEQ ID NO: 17)CD127 R: CGT CCA TTT GTT TTC ATC CTT T

Power SYBR Green PCR Master Mix was from Applied Biosystems. Triplicatesamples of cDNA were subjected to 40 cycles of amplification in an ABIPrism 7900 instrument, according to the manufacturer's protocol. mRNAexpression, normalized to endogenous β-actin expression, was quantifiedby the comparative critical threshold (Ct) method according to theformula 2-ΔΔCt, as described in the ABI User Bulletin 2(docs.appliedbiosystems.com/pebiodocs/04303859.pdf).

Results

Consistent with the flow cytometric expression analysis, CD52, CD127 andGITR transcripts were higher in CD52^(hi) cells than CD52^(lo) cells(FIG. 5).

Example 6 Suppression by CD52^(hi) Cells is not Influenced by the Levelof Expression of CD24 Methods

In order to analyze expression of the structurally related CD24GPI-anchored glycoprotein, CFSE-labelled PBMCs were incubated with TTfor 7 days and sorted into CD52^(hi)CD24^(lo), CD52^(hi)CD24^(hi),CD52^(lo)CD24^(lo) and CD52^(lo)CD24^(hi) CD4⁺ T-cells. Each population(5,000 cells) was incubated with sorted CD52^(lo) responder cells(5,000) and irradiated PBMCs (20,000) and analysed by ELISpot assay.Results are mean+sem of triplicates.

Results

Expression of the GPI-anchored glycoprotein, CD24, structurally relatedto CD52 (Tone et al, 1999), was higher on CD52^(hi) CD4⁺ T-cells.Although antigen-activated CD24^(hi) CD4⁺ T-cells, unlike CD52^(hi) CD4⁺T-cells, were not suppressive it was important to determine if CD24better delineated CD52^(hi) CD4⁺ T-cells with suppressor function.TT-activated PBMCs were sorted into four distinct CD4⁺ populationsaccording to both CD52 and CD24 expression and then tested forsuppressor function after re-activation with TT. This revealed thatsuppression by CD52^(hi) cells was not influenced by expression of CD24(FIG. 6).

Example 7 CD52^(hi) Treg Function Does Not Require Cell-Cell ContactMethods

³H-thymidine uptake (cpm) by TT-activated and sorted CD52^(hi) andCD52^(lo) CD4⁺ cells either combined or separated by a semi-permeable0.4 μm transwell and re-activated with TT. CFSE-labelled PBMCs wereincubated with TT for 7 days and sorted into CD52^(hi) and CD52^(lo)CD4⁺ cells. Sorted cells (100,000 each) were incubated with irradiatedautologous PBMCs (400,000) and TT in 48-well plates; in the presence ofthe transwell both compartments contained irradiated PBMCs and TT.³H-thymidine uptake by cells in the bottom compartment was measuredafter 48 hrs.

Results

The suppressor function of antigen-activated CD52^(hi) CD4⁺ T-cells wasretained across a transwell without cell-cell contact (FIG. 7). Thus,the present disclosure demonstrates that CD52^(hi) CD4⁺ Treg suppressionis mediated at least in part by a soluble mediator. As discussed inVignali et al. (2008), inhibitory cytokines have previously beeninvestigated as possible soluble mediators of Treg suppression, thoughresults have been inconclusive and the general perception has remainedthat cell-cell contact is essential for Treg suppressor function. Theresults disclosed herein suggested that CD52^(hi) CD4⁻ T-cells eitherremoved a soluble factor required for the function of responder T-cellsor produced a soluble factor that suppressed responder T-cells.

Example 8 Analysis of IL-2 in CD52^(hi) Treg Function Methods

The role of IL-2 was investigated in a number of experiments includingthe use of quantitative real time RT-PCR to determine expression levels.In the quantitative RT-PCR analysis, total RNA was extracted from cellswith the RNAeasy Mini Kit (Qiagen, Melbourne, Australia), treated withRNase-free DNase (Qiagen) and quantified with the Agilent 2100Bioanalyser. cDNA was reverse transcribed from 10 ng RNA/reaction.Primers for PCR, designed with PrimerExpress software and synthesized bySigma-Aldrich (Castle Hill, NSW, Australia), were:

(SEQ ID NO: 18) IL-2α ′5 TACAGGATGCAACTCCTGTCTT, (SEQ ID NO: 19)′3 GCTCCAGTTGTAGCTGTGTTTT; (SEQ ID NO: 20) IL-27β′5 GCTGTTCTCCATGGCTCCCTAC, (SEQ ID NO: 21) ′3 GTCGGGCTTGATGATGTGCT;(SEQ ID NO: 22) IL-12α ′5 CTCCAGAAGGCCAGACAAACTC, (SEQ ID NO: 23)′3 CCAATGGTAAACAGGCCTCCAC.

Power SYBR Green PCR Master Mix was from Applied Biosystems. cDNA wassubjected to 40 cycles of amplification in an ABI Prism 7900 instrument,according to the manufacturer's protocol. mRNA expression, normalized toendogenous β-actin expression, was quantified by the comparativecritical threshold (Ct) method according to the formula 2-ΔΔCt, asdescribed in the ABI User Bulletin 2(docs.appliedbiosystems.com/pebiodocs/04303859.pdf).

Results

Consumption or degradation of IL-2 by CD52^(hi) CD4⁺ T-cells wasconsidered an unlikely mechanism of suppression for several reasons: i)exogenous IL-2 did not overcome suppression (FIG. 2C); ii) quantitativeRT-PCR revealed that IL-2 gene expression was actually higher inCD52^(hi) cells; thus, 24 h after re-activation by GAD65 the expressionof IL-2α mRNA in CD52^(hi) relative to CD52^(lo) cells was 1.54±0.15(mean±sem, n=3); iii) IL-2 concentration in the medium of CD52^(hi)cells was higher than for CD52^(lo) cells, both resting (89.5±4.82 v64.9±3.10 pg/ml) and after re-activation with GAD65 (138.7±4.16 v82.4±1.78 pg/ml) (mean±sem, n=3; P=0.02, Kruskal-Wallis test); iv) inthe media in which IL-2 was measured, soluble IL-2 receptor-α (CD25) wasundetectable (data not shown). Thus, the removal of IL-2 was thought tobe an unlikely mechanism of CD52^(hi) Treg suppression.

Example 9 Analysis of Other Putative Mediators of CD52^(hi) TregFunction

Treg suppression was then found to be unchanged in the presence ofagents that block the action or production of factors reported tomediate suppression by CD4⁺ Treg cells (Sakaguchi et al., 2008, 2009;Shevach, 2006, 2009; Vignali et al., 2008). These included neutralizingmonoclonal antibodies to IL-10Rα or TGF-βRII singly or in combination(10 m/ml each), the cyclooxygenase-2 (COX-2) inhibitor indomethacin (20μM) (which blocks prostaglandin E2 production), the pan nitric oxidesynthase inhibitor N(G)-monomethyl-L-arginine (800 μM) (which blocksnitric oxide production), the indoleamine-2,3-dioxygenase (IDO)inhibitor 1-methyl-dl-tryptophan (200 μM) (which blocks production ofinhibitory tryptophan metabolites) and the adenosine A2A receptorantagonist SCH58261 (20 μM) (which blocks the action of adenosine) (datanot shown). Recently, a novel suppressor cytokine, IL-35, a heterodimerof IL-27β (EBi3) and IL-12α (p35) subunits, was shown to be secreted byCD4⁻CD25⁺ Treg cells (which also required cell-cell contact forsuppression) (Collison et al., 2007). IL-35 was unable to be measureddirectly because antibodies to IL-35 or its receptor were not available.However, 24 h after re-activation by GAD65 the expression of IL-27β andIL-12α mRNA was lower in CD52^(hi) than CD52^(lo) CD4⁺ cells(0.423±0.188 vs 1.38±0.224; mean±sem, n=3), indicating that IL-35 isunlikely to account for suppression by CD52^(hi) CD4⁺ T-cells.

Example 10 Soluble CD52 is a Mediator of CD52^(hi) Treg SuppressionMethods

CFSE-labelled PBMCs were incubated with GAD65 for 7 days and sorted intoCD52^(hi) and CD52^(lo) CD4⁺ T-cells. Sorted cells were re-activatedwith GAD65 and media collected after 24 hrs. Media were concentrated10-fold, fractionated by SDS-PAGE, transferred to a PDVF membrane andblotted with a rabbit polyclonal antibody to CD52 in order to detect thepresence of soluble CD52 in the media.

The phospholipase C inhibitor U73122 was then analysed as a potentialinhibitor of soluble CD52 production. CFSE-labelled PBMCs were incubatedwith TT for 7 days and sorted into CD52^(hi) CD4⁺ T-cells. Sorted cellswere re-activated with TT and media collected after 24 hrs and subjectedto immunoblotting as above. Separately, CFSE-labelled PBMCs wereincubated with TT for 7 days and sorted into CD52^(hi) and CD52^(lo)CD4⁺ T-cells, which then were incubated together (5,000 of each) inELISpot plates with irradiated PBMCs (20,000) and TT±phospholipase Cinhibitor U73122. Results are mean+sem of triplicates.

In addition, antibody to the carbohydrate moiety of CD52 was analysed asanother potential inhibitor of suppression by TT-activated CD52^(hi)CD4⁺ T-cells. Procedures were as described for the phospholipase Cinhibitor U73122 above except that cells in the ELISpot assay wereincubated with or without TT and either 10 μg/ml anti-CD52 (CF1D12) orisotype control (IgG3) monoclonal antibody. Results (mean±sem) arerepresentative of three independent experiments.

Results

Immunoblotting revealed that CD52 was present in the medium of CD52^(hi)CD4⁺ T-cells that had divided in response to GAD65, and increased inquantity after their re-activation by GAD65 (FIG. 8A). The same resultwas found with TT as antigen and the phospholipase C inhibitor, U73122,added before re-activation with TT reduced the quantity of CD52 in themedium (FIG. 8B). Moreover, inhibition of phospholipase C reversedsuppression by CD52^(hi) CD4⁺ T-cells in a dose-dependent manner (FIG.8C). The monoclonal antibody CF1D12, which interacts with the terminalcarbohydrate on the CD52 peptide (Hale, 2001), prevented suppression byCD52^(hi) of CD52^(lo) CD4⁺ T-cells (FIG. 8D). Together, these findingsindicated that suppression by CD52^(hi) CD4⁺ T-cells was due to solubleCD52, released by phospholipase cleavage in response to stimulation byantigen.

Example 11 Further Analysis of Soluble CD52 Effector Function

In considering a more abundant source of native soluble CD52 it waspostulated that CD52 might be released spontaneously from some celllines, such as the Daudi B lymphoblast cell line in which GPIbiosynthesis is defective due to a deficiency of the PIGY gene product(Hu et al., 2009).

Methods

Media from sorted CD4⁺CD52^(hi) and CD52^(lo) cells were collected 24hrs after re-activation of cells with GAD65 or TT. Media from cell lines(Daudi, Raji, Jurkat and K562) were collected and concentrated 10-foldby freeze-drying. Samples were fractionated by SDSPAGE and transferredto a PVDF membrane. After blocking with 5% non-fat milk the membrane wasincubated with rabbit polyclonal antibody to CD52 (1 ug/mL), washed,incubated with goat anti-rabbit IgG-horseradish peroxidase antibody andvisualized by enhanced chemiluminescence.

Separately, PBMCs (200,000 cells) were cultured for 7 days in IMDMcontaining 20% Daudi cell conditioned medium with TT and eitheranti-CD52 (CF1D12) or isotype control antibody (10 μg/mL). To depletesoluble CD52, Daudi medium was incubated overnight with rabbit anti-CD52polyclonal antibody (1 μg/ml medium) followed by precipitation withprotein G-Sepharose for 1 h at 4° C. Results (mean±sem) arerepresentative of three independent experiments.

Results

Screening several cell lines revealed the presence of CD52 in culturemedia of Daudi and K562 cells (FIG. 9A), Daudi medium suppressedTT-activated proliferation of PBMCs and suppression was reversed eitherby CF1D12 antibody or by immunodepletion (confirmed by immunoblotting)of CD52 (FIG. 9B), demonstrating that T-cell suppression was due to CD52in the medium. CD52 was soluble and not present in exosomes or membraneparticles because suppression was unaffected by centrifuging the mediumat 100,000×g for 30 min (data not shown).

Example 12 Replication of Soluble CD52 Effector Function with CD52-FcMethods

To further explore the immunosuppressive function of soluble CD52,mature cell surface CD52 was cloned in a lentivirus vector as a fusionprotein in-frame with the Fc fragment of immunoglobulin G and aC-terminal Strep-tag sequence for purification. An Fc only construct wascloned as a control. Constructs were expressed stably in Daudi cells ortransiently in HEK293T cells and soluble recombinant proteins purifiedfrom medium by elution with desthiobiotin from Streptactin resin.

The scheme for constructing DNAs encoding fusion proteins is shown inFIG. 10. A mutated human IgG1 Fc fragment (Armour et al., 2003) joinedto the signal peptide (SigP) sequence of CD52 was generated by PCR. Thisincluded a flexible GGSGG linker and two cleavage sites for Precissionand Factor Xa proteases between the SigP and Fc fragment, and aStrep-tag II sequence for purification (Schmidt and Skerra, 2007) at theterminus of the Fc fragment. Primers, as designated in FIG. 10, used togenerate and clone Fc constructs, were:

1F1:  (SEQ ID NO: 24) GAAGTTCTGTTCCAGGGGCCCATCGAAGGTCGTGGTG; 1R1: (SEQ ID NO: 25) TCATTTTTCGAACTGCGGGTGGCTCCAGGCGCTTTTACCCGGAGACAG; 1F2: (SEQ ID NO: 26) GGGGGTTCCGGGGGACTGGAAGTTCTGTTC; 1R2:  (SEQ ID NO: 27)CTTGATATCGAATTCTCATTTTTCGAACTG; 2F:  (SEQ ID NO: 28)CGCTGTTACGGATCCCCACCATGAAGCGCTTCCTC; 2R1:  (SEQ ID NO: 29)TCCACCGCTACCTCCTGAGGGGCTGCTGGT; 2R2:  (SEQ ID NO: 30)TCCACCGCTACCTCCTGAGAGTCCAGTTTG.

A CD52-Fc construct comprising the CD52 SigP and extracellular domain(ECD) joined to the Fc fragment was generated by PCR. Primers used were:2F, 2R1, 1F2 and 1R2. PCR products were digested with BamHI/EcoRI andligated into the FTGW lentivirus vector (Herold M J et al., 2008).Clones were also verified by sequencing. Lentivirus particles wereproduced by CaPO4-mediated transfection of HEK293T cells seeded in 6 cmdishes with 10 ug of vector DNA together with three helper plasmids(pMDLRRE, pRSV-REV, and pVSV-g). Virus-containing cell culture mediumwas collected 48 hrs after transfection and passed through a 0.45 μmfilter. One milliliter was used to transduce 1×10⁶ Daudi cells grown inDME media supplemented with 10% FCS, 100 mM non-essential amino acids, 2mM glutamine and 5×10⁻⁵ M 2-mercaptoethanol. Cells were screened for thehighest expression of protein by intracellular staining and flowcytometry. CD52-Fc or Fc control proteins were purified from medium bysingle-step affinity chromatography on Streptactin resin and elutionwith 2.5 mM desthiobiotin in 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH8.0, as per the manufacturer's instructions. After dialysis, SDS-PAGErevealed single Coomassie blue-stained bands of predicted size whosespecificity was confirmed by Western blotting.

Assays for Effects of Recombinant Fc Fusion Proteins

PBMCs (2×10⁵ cells/well) or purified CD4⁺ T-cells (5×10⁴ cells/well) incomplete IMDM medium-5% heat-inactivated pooled human serum wereincubated in round-bottomed 96-well plates with or without 10 Lfu/ml TTand different concentrations of CD52-Fc or Fc proteins, in a totalvolume of 200 μl, at 37° C. in 5% CO₂-air for up to 7 days. ³H-thymidine(1 μCi/well) was added and after a further 18 h cells were harvested andradioactivity incorporated into DNA was measured by scintillationcounting. Medium was sampled for assay of cytokines after 48 hrincubation. Dendritic cells (DCs) were isolated from PBMCs as described(Mittag et al., 2011). In brief, PBMCs were first enriched for DCs bymagnetic bead depletion of cells labelled with antibodies to lineagemarkers (CD3, CD19, CD56). Cells were then stained with fluorescentantibodies to HLA-DR, CD11c, CD1b/c, CD304 and CD14 and flow sorted topurify CD1b/c+HLA-DR+CD11c+ conventional DC, CD304+HLA-DR+CD11c−plasmacytoid DC and CD14+CD16-CD11c+ monocytes. Purified DCs werepre-incubated with CD52-Fc or Fc protein at 3.3 μM for 30min at 37° C.and washed twice. They were then serially diluted from 6000 cells/wellin a 96-round bottom well plate and incubated with CF SE-labelled CD4⁺T-cells (5×10⁴/well) isolated from a different donor. After 6 days, theallogeneic T-cell response was measured as frequency of dividingCFSE^(lo) cells determined by flow cytometry.

As described above, PBMCs (200,000) were cultured with TT for 7 days andpurified CD4⁺ T-cells (20,000) with anti-CD3 (100 ng/ml) and anti-CD28(200 ng/ml) antibody for 48 hr, with 4 times the number of irradiatedPBMCs in 200 μl round bottom wells, in the presence of recombinantCD52-Fc or Fc protein control protein at the indicated concentrations.³H-thymidine uptake was measured over the final 16 h of incubation.Results (mean±sem of triplicates) are representative of six independentexperiments.

Media from PBMCs activated with TT±3.3 μM CD52-Fc or Fc proteins weresampled after 48 h incubation and assayed for cytokines by multiplexbead array.

CD52-Fc (20 μg) was incubated with or without PNGase F (1,000 units) in20 μl PBS overnight at 37° C. in order to cleave N-linked carbohydrate,and the reaction terminated by heating at 75° C. for 10 min.Specifically, PNGase F cleaves asparagine-linked oligosaccharidesbetween two N-acetylglucosamine subunits immediately adjacent to theasparagine residue to generate a truncated carbohydrate with oneN-acetylglucosamine residue remaining on the asparagine. PBMCs wereincubated with TT and treated or untreated CD52-Fc (final 2.5 μM) for 7days at 37° C., and 3H-thymidine uptake then measured as above.

Results

With PBMCs, the proliferative response of T-cells to TT was suppressedby CD52-Fc in a dose-dependent manner (FIG. 11A), and CD52-Fc suppressedthe secretion of cytokines typifying different T-cell lineages (FIG.11C). The effect of CD52-Fc on T-cell function was direct because itsuppressed proliferation of purified CD4⁺ T-cells in response to T-cellreceptor cross-linking with anti-CD3 antibody and co-stimulation withanti-CD28 antibody (FIG. 11B). Evidence that CD52-Fc did not requireantigen-presenting cells for T-cell suppression was obtained by showingthat exposure of purified dendritic cells to CD52-Fc did not affecttheir ability to elicit an allogeneic T-cell response (FIG. 13).

As shown (FIG. 8D), the ability of the CF1D12 antibody to blocksuppression by native CD52 implied that suppression may be mediated bythe carbohydrate moiety of CD52. To examine its role in recombinantCD52-Fc, the N-linked carbohydrate was cleaved with the endoglycosidasePNGase F. This reduced the molecular weight of CD52-Fc from ˜48 to ˜30kDa as predicted from loss of the carbohydrate and reduced itssuppressive effect (FIG. 11D), confirming the role of the carbohydratemoiety in mediating the suppressive effect of soluble CD52.

Example 13 Further Analysis of CD52 Carbohydrate Function Methods

To further explore the role of the CD52 carbohydrate moiety in mediatingT-cell suppression, CD52-Fc (3.3 μM) was incubated with neuraminidase (1unit) or carrier buffer only in 20 μl for 30 min at 37° C., asrecommended by the supplier. PBMCs were then incubated withTT±neuraminidase-treated or untreated CD52-Fc (final 3.4 μM) in anELISpot plate and developed after 24 h at 37° C. for IFN-γ spots.

Separately, PBMCs were incubated in an ELISpot plate with TT and CD52-Fc(3.4 μM) and different concentrations of affinity-purified goat antibodyto the extracellular domain of Siglec-10, or Fc (3.4 μM)±antibody, ordifferent concentrations of recombinant Siglec-10-Fc, beforenon-adherent cells were transferred to an ELISpot plate for 24 hrsbefore development of IFN-γ spots.

In order to investigate the possibility that soluble CD52 may act viaother Siglec receptors than Siglec-10, CD4⁺ T-cells (20,000) wereincubated in triplicate ELISpot plate wells at 37° C. with TT, togetherwith CD52-Fc or Fc (3.4 μM each) and anti-human Siglec antibodies (10μg/ml each) or recombinant human Siglec 2-Fc (20 μg/ml), as indicated inFIG. 12E. After 20 h, wells were washed and developed for IFN-γ spots.

Results

Treatment with neuraminidase to remove terminal sialic acids reducedsuppression by CD52-Fc (FIG. 12A). The complex polylactosamine structureof the CD52 carbohydrate is proposed to terminate in α2-6 and possiblyα2-3 sialic acids decorating galactose in β1-4 linkage withN-acetylglucosamine (Treumann et al., 1995). This sialoside sequence isrecognized by human sialic acid binding Ig-like lectin-10 (Siglec-10), acell surface transmembrane receptor and immunoglobulin superfamilymember bearing two cytoplasmic immunoreceptor tyrosine-based inhibitionmotifs (ITIMs) (Munday et al., 2001; Crocker et al., 2007). AlthoughSiglec-10 has not been detected on mouse T-cells (Crocker et al., 2007)and some other Siglecs are not expressed on human T-cells (Nguyen etal., 2006) we found that Siglec-10 was expressed on human CD4⁺ T-cellsand was upregulated by activation (FIG. 12B). Notably, suppression ofT-cell function by CD52-Fc was reduced either by antibody to theextracellular domain of Siglec-10 (FIGS. 12C, 12E) or by solublerecombinant Siglec-10-Fc (FIG. 12D). The same concentrations ofSiglec-10-Fc also reduced suppression by CD52^(hi) CD4⁺ T-cells (datanot shown), indicating that both recombinant and native CD52 recognizeSiglec-10. T-cell suppression by CD52-Fc was not reduced to the sameextent by antibodies to other Siglecs than Siglec-10 or by recombinanthuman Siglec 2-Fc. These findings show that suppression by CD52 could beaccounted for at least in part by its interaction with Siglec-10.

Example 14 CD52^(hi) T-Cells Protect Against Autoimmune DiseaseMaterials and Methods Mice

C57/B16, NODLt and RIP.B7/NODSCID mice were bred and maintained at theWalter and Eliza Hall Institute of Medical Research. OVA-specific classI restricted TCR transgenic mice (Hogquist et al., 1993) andOVA-specific class II restricted TCR transgenic mice (Barnden et al.,1998) have been previously described. Foxp3^(GFP) reporter mice wereprovided by Dr Yifan Zhang.

Reagents, Antibodies and Flow Cytometry

Cells were cultured in RPMI media supplemented with 10% FCS, 1:100GIBCO™ GlutaMAX™-I Supplement (Invitrogen), 1:1000 2-mercaptoethanol(Sigma), 1:100 NEAA (gibco). Monoclonal anti-CD52 antibodies wereobtained from MBL International, clone BTG-2, PE conjugation orunlabeled. Polyclonal anti-CD52 antibody obtained from Santa CruzBiotechnology, Inc (sc27555) was used for Western Blot analysis.Monoclonal anti-CD4 (L3T4, clone GK1.5) and anti-CD8a (Ly-2, clone53-6.7) antibodies were obtained from eBiosciences. Anti-CD25 (clone3c7) was obtained from BioLegend. CD3-FITC antibody, FoxP3 staining kitwas obtained from eBioscience. Anti-CD3 (clone 2c11), anti-CD28 (clone37.51) and isotype control monoclonal antibodies were from WEHIMonoclonal Antibody Lab. Flow cytometric analyses were done on aFACSAria with the FACS Diva software. Cells were sorted with a MoFlowcell sorter (Cytomation, Fort Collins, Colo.).

Cell Isolation

Spleens were harvested and passed through a 70 μm mesh, treated witherythrocyte lysis buffer and washed. For activation of cells,splenocytes were cultivated on plate bound anti-CD3 (2 μg/ml) plussoluble anti-CD28 (1 μg/ml) for 3 days. OTI or OTII splenocytes wereincubated with 0.5 μg/ml OTI or 5 μg/ml OTII peptide for 4 days beforeanalysed. For cell sorting experiments, naive or activated splenocytesfrom C57/B16, OTI or OTII, NODLt or Foxp3-GFP mice were labelled witheither CD3-FITC (eBioscience), CD4-APC (eBioscience), CD8-APC(eBisoscience) and CD52-PE (MBL International). Labelled cells wereseparated with a MoFlow Cytometer and purity was ˜95%. Isolated cellswere either used for RNA purification, for T-cell proliferation assaysor in vivo experiments.

Proliferation Assays

Sorted naive or activated CD4⁺CD52^(hi) or CD8⁺CD52^(hi) T-cells (2×10⁴,in transwell experiments 1×10⁵) were cultured with CD4⁺CD52^(lo) orCD8⁺CD52^(lo) T-cells at a ratio of 1:1 and stimulated with 1 μg/mlsoluble anti-CD3 (2c11) plus (8×10⁴, in transwell experiment 4×10⁵)irradiated T-cell depleted APCs (2000rad irradiation dose). 0.4 μMtranswells (Corning, polycarbonate membrane transwell inserts Cat No3413) were placed in between cells. In blocking experiments, 15 μg/mlanti-CD52 (rat IgG2a, MBL International) or isotype control was added.Proliferation assays were performed for 72 h in 96 well round bottomplates in a final volume of 200 μl RPMI medium that contained 10% fetalcalf serum. 1 μCi/well [³H] thymidine was added for the last 10 hours ofthe experiment and thymidine incorporation was measured by scintillationcounting. Alternatively, CFSE labelled responder cells were used asreadout for proliferation. Naive splenocytes or CD4⁺CD52^(lo) orCD8⁻CD52^(lo) T-cells were resuspended in warm PBS+0.1% BSA at a cellnumber of 10×10⁶ per ml. 5 μM CFSE was added and quickly resuspended.Cells were incubated at 37 degrees for 5 min before washed 3 times withcold buffer containing at least 10% BSA or FCS. CFSE labelled respondercells were incubated with CD4⁺CD52^(hi) or CD8⁺CD52^(hi) T-cells (plusadditional controls) for up to 7 days and analysed using the FACS Aria.

Two Colour Assay

CD4⁻CD52^(hi) or CD8⁺CD52^(hi) T-cells were stained with the celldivision marker PKH.26 (Sigma) according to manufacturersrecommendations. Briefly, up to 1×10⁷ cells were resuspended in DiluentC (provided by the kit) and mixed with 2 μM PKH.26 for 4 min at roomtemperature. Cells were washed 3 times with buffer containing at least10% FCS. Responding CD4⁺CD52^(lo) or CD8⁺CD52^(lo) T-cells were stainedwith CFSE as described above. Cells were cultivated alone or togetherfor 4-6 days.

Realtime RT-PCR

Total RNA was prepared from sorted T-cells using the RNeasy kit fromQiagen. The cDNA was synthesized using oligo-dT primers (Qiagen, 0.4μg/μl) and M-MLV reverse transcriptase (4000U, Applied Biosystems),following the manufacturers recommendations. Realtime RT-PCR wasperformed in an ABI PRISM 7900 cycler (Applied Biosystems) using aQuantitect SYBR Green PCR Kit (Qiagen, Cat No 204143) and specificprimers optimised to amplify 100-150 bp fragments of different genes. Athreshold was set in the linear part of the amplification curve and thenumber of cycles needed to reach the threshold was calculated for everygene. Relative mRNA expression was determined by normalization to areference gene (b-Actin or RPS9).

Primer sequences are:

CD52 (SEQ ID NO: 31) FORW-GTT GTG ATT CAG ATA CAA ACA GGA (SEQ ID NO: 32) REV-AGG TAT TGG CAA AGA AGA GGA A  IL-2 (SEQ ID NO: 33)FORW-TCA AGC TCC ACT TCA AGC TCT AC  (SEQ ID NO: 34)REV-CCT GTA ATT CTC CAT CCT GCT C  IL-4 (SEQ ID NO: 35)FORW-TGA GAG AGA TCA TCG GCA TTT T  (SEQ ID NO: 36)REV-CTC TCT GTG GTG TTC TTC GTT G  IL10 (SEQ ID NO: 37)FORW-TCG GAA ATG ATC CAG TTT TAC C  (SEQ ID NO: 38)REV-ATC CTG AGG GTC TTC AGC TTC  IL-13 (SEQ ID NO: 39)FORW-GAG-CTG-AGC-AAC-ATC-ACA-CAA  (SEQ ID NO: 40)REV-AATCCAGGGCTACACAGAACC FoxP3 (SEQ ID NO: 41)FORW-ATG-TTC-GCC-TAC-TTC-AGA-AAC-C  (SEQ ID NO: 42)REV-CAA-ATT-CAT-CTA-CGG-TCC-ACA-C  CD127 (SEQ ID NO: 43)FORW-GCC CAC CAG AAA CAG TTA GAA G  (SEQ ID NO: 44)REV-AGT CAG GGG ACC TAG AGG AAA G  CTLA-4 (SEQ ID NO: 45)FORW-AGT TTC CTG GTC ACT GCT GTT T  (SEQ ID NO: 46)REV-TTT TCA CAT TCT GGC TCT GTT G  FASLG (SEQ ID NO: 47)FORW-CGG-TGG-TAT-TTT-TCA-TGG-TTC-T  (SEQ ID NO: 48)REV-TGA-TAC-TTT-AAG-GCT-TTG-GTT-GG  TGFb1 (SEQ ID NO: 49)FORW-TAT TGC TTC AGC TCC ACA GAG A  (SEQ ID NO: 50)REV-CAG ACA GAA GTT GGC ATG GTA G  TGFb2 (SEQ ID NO: 51)FORW-TAA GAG GGA TCT TGG ATG GAA A  (SEQ ID NO: 52)REV-CTG AGG ACT TTG GTG TGT TGA G  IFNg (SEQ ID NO: 53)FORW-CAA-AAG-GAT-GGT-GAC-ATG-AAA-A  (SEQ ID NO: 54)REV-TTG CTG TTG CTG AAG AAG GTA G  IL-12alpha (SEQ ID NO: 55)FORW-TCA CGC TAC CTC CTC TTT TTG G  (SEQ ID NO: 56)REV-CAT CTG TGG TCT TCA GCA GGT TT  Ebi3 (SEQ ID NO: 57)FORW-CCT TCC CGG ACA TCT TCT CTC T  (SEQ ID NO: 58)REV-GCA ATA CTT GGC ATG GGG TTT  RARA (SEQ ID NO: 59)FORW-GGA CAA GAA CTG CAT CAT CAA C  (SEQ ID NO: 60)REV-GCT TGG GTG CCT CTT TCT TC  GITR (SEQ ID NO: 61)FORW-CCT-AGG-TCA-GCC-GAG-TGT-AGT-T  (SEQ ID NO: 62)REV-CAC-ATA-TGC-ACC-TTT-CTT-TTG-G  GRANZMB (SEQ ID NO: 63)FORW-TCC TTA TTC GAG AGG ACT TTG TG  (SEQ ID NO: 64)REV-CTG GGT CTT CTC CTG TTC TTT G  ALDH1A2 (SEQ ID NO: 65)FORW-ACA GGA GAG CAA GTG TGT GAA G  (SEQ ID NO: 66)REV-TCC ACA CAG AAC CAA GAG AGA A  ACTIN (SEQ ID NO: 67)FORW-GAT CTG GCA CCA CAC CTT CT  (SEQ ID NO: 68)REV-GGG GTG TTG AAG GTC TCA AA Adoptive Transfer of CD52^(hi) Depleted Splenocytes into NOD Recipients

CD52^(hi) depleted total splenocytes or splenocytes depleteted of CD3⁺,CD4⁺ or CD8⁺CD52^(hi) T-cells were injected iv into recipient mice.Recipient mice were either irradiated male NOD mice (8 week old male NODmice, 750 rad irradiation dose, 4 hours before transfer of 1 to 1.2×10⁷cells) or 8-week old RIP.B7/NOD.SCID mice, receiving 2×10⁶ cells. Micewere monitored for signs of diabetes measuring urine glucose 3 times aweek using Diastix from Bayer. If urine glucose exceeds 20 mM, bloodglucose is measured. Mice are designated diabetic if consecutive bloodglucose readings are above 20 mM glucose.

Insulitis Score

4 weeks postadoptive transfer of cells mice were sacrificed. Pancreatawere harvested and fixed overnight in Bouin's solution and thentransferred to 70% ethanol. Fixed pancreata were embedded in paraffinblocks, a minimum of 12 8-μm sections were cut at least 150 μm apart.The sections were stained with haematoxylin-eosin and evaluated forincidence and severity of insulitis in light microscopy independently bytwo investigators. A minimum of 10 islets from each mouse were observedand the degree of mononuclear cell infiltration was scored using thefollowing ranking: 0=no infiltration; 1=peri-ductal infiltrate;2=peri-islet infiltrate; 3=intra-islet infiltrate; 4=beta celldestruction.

Results

Transfer of CD52^(hi)-depleted splenic lymphocytes from 8 week-old NODmice into NOD.scid mice lead to rapid onset of diabetes; non-depletedcells had no effect (FIG. 14). Transfer of CD52^(hi)-depleted CD3⁺T-cells accelerated diabetes onset, but was not as efficient as thedepletion of total lymphocyte CD52^(hi) cells (FIG. 15). Thus, CD52^(hi)lymphocytes were shown to protect against autoimmune diabetes.

Example 15 The Frequency of CD52^(hi) CD4⁺ T-Cells Generated in Responseto Simulation by GAD65 is Impaired in Type 1 Diabetes Methods

PBMCs stained with CFSE were cultured with GAD65 or TT for 7 days beforedetermination of CD52^(hi) CD4⁺ T-cell frequency by flow cytometricanalysis.

Results

Individuals with and at risk for type 1 diabetes have fewer CD52^(hi)CD4⁺ T-cells than healthy individuals in response to GAD65 but not TT(FIG. 16). The horizontal bar is the median for each group. Overall Pvalues for analysis of variance were determined by the Kruskal-Wallistest; Dunn's multiple comparison test then revealed significantdifferences between both Pre-T1D and T1D compared Healthy or T2D atP<0.05.

Example 16 T-Cell Suppression by CD52^(hi) CD4⁺ Cells Generated inResponse to GAD65 is Impaired in Pre-Clinical Type 1 Diabetes Methods

CFSE-labelled PBMCs from individuals with islet cell autoantibodies atrisk for type 1 diabetes were incubated with GAD65 for 7 days and sortedinto CD52^(hi) and CD52^(lo) CD4⁺ T-cells according to the methodsdescribed herein. Sorted cells (5,000) were incubated in ELISpot plateswith irradiated PBMCs (20,000).

Results

As shown in FIG. 17, suppressor function of CD52^(hi) CD4⁺ cellsgenerated in response to GAD65 is impaired in comparison to suppressorfunction of CD52^(hi) CD4⁺ cells generated in response to TT. Resultsare representative of 6 at-risk subjects. Thus, CD52^(hi) CD4⁺ cellsuppressor function is impaired in pre-clinical T1D.

Example 17 Soluble CD52 Dramatically Reduces Blood Glucose Levels in NODMice Methods

Female NOD mice were monitored by weekly testing for urine glucose anddiabetes was diagnosed in mice with a positive urine test by a bloodglucose concentration>14 mM. As soon as hyperglycemia was confirmed micewere given either CD52-Fc or Fc, 20 μg i.p., six doses on alternatedays, and their blood glucose concentrations then monitored twiceweekly.

Results

Soluble CD52-Fc was shown to reduce blood glucose levels (FIG. 18). Asshown, administration of CD52-Fc had a rapid and significant effect toreduce blood glucose levels, demonstrating the suitability of solubleCD52 as a therapeutic for the treatment of autoimmune diseases such astype 1 diabetes.

Example 18 Development of Diabetes in NOD.SCID Mice After Transfer fromDiabetic NOD Mice of Splenocytes Treated Ex Vivo with hCD52-Fc or FcMethods

5×10⁶ rhCD52 Fc- or Fc-treated diabetic NOD splenocytes were injectedinto NOD.SCID mice. Splenocytes from female diabetic mice were isolatedand incubated with either 50 ug/ml recombinant human CD52-Fc or Fcprotein for 1.5 hr in ‘CD52 buffer’ (Tris buffered saline+2 mM of MgCl2,CaCl2 and MnCl2+5 mM glucose+1% mouse serum). Cells were re-suspended inPBS and 1×10⁷ cells were injected into male NOD.SCID mice (12 pergroup).

Results

Treatment of splenocytes from diabetic NOD mice ex vivo with CD52-Fcresulted in an increase in the diabetes-free survival in NOD.SCID miceinto which the treated splenocytes were implanted (FIG. 19). This is yetfurther evidence of the therapeutic use of soluble CD52 for thetreatment of autoimmune diseases such as type 1 diabetes.

Example 19 Human CD52-Fc Suppresses Mouse OT-II Cells Methods

Mouse ovalbumin (Ova)-specific TCR transgenic CD4 (OT-II) T-cells are aconvenient model for testing immune suppression since approximately halfof the CD4 T-cells are specific for ovalbumin and T-cell responses aretherefore strong and predictable. Splenocytes (1×10⁵) from 10 week-oldfemale OT-II mice were incubated for 3 days in round bottom 96-wellplates in 200 ml RPMI-1640 medium containing 5% FCS and theconcentrations indicated in FIG. 20 of ova protein or peptide, oranti-CD3 antibody (clone 2C-11), and recombinant human CD52-Fc or Fcprotein. ³H-thymidine uptake was measured over the last 16 h of culture.Results are mean±sem of triplicates.

Results

As shown in FIG. 20, CD52-Fc significantly reduced T-cell proliferationin response to stimulation by ova protein or peptide in a dose-dependentmanner, providing further evidence of the therapeutic potential ofsoluble CD52 in treating autoimmune diseases.

Example 20 Seminal Fluid-Derived Soluble CD52 Suppresses Human T-CellProliferation Methods

CD52 was identified in human semen samples using the following ELISAprotocol. Initially, seminal fluid (SF) was centrifuged at 500 g for 5min to pellet sperm, confirmed by microscopic inspection of thesupernatant. Anti-human CD52 antibody (Biolegend #338202) was used asthe capture agent (1:100 in PBS; 50 μl/well overnight; 4° C.). Wellswere washed 3 times in PBS-0.01% Tween, followed by 3 times in PBS. Asolution of 5% BSA/PBS (BSA Sigma A7906) was used to block wells (200μl/well, 1 hr at room temperature (RT)). Washing was performed as above.Blank wells were included as controls. Semen samples were diluted in 5%BSA/PBS and added at 50 μl/well. Samples were incubated in the wells for3 hr at RT. Washing was performed as above. For detection, CampathmAb-HRP was used at 1:1000 in 5% BSA/PBS (100 μl/well; 1 h at RT).Washing was performed as above. 3,3′,5,5′-Tetramethylbenzidine (TMB) wasadded and the plates read at 450 nm.

CD52 immunodepletion was performed according to the following protocol.200 μl Protein G-Sepharose was aliquoted into 2 Eppendorf tubes,followed by washing ×2 with 1 ml PBS, the supernatant being discarded. 5mg Campath mAb was added to one Eppendorf and 5 mg ‘Octagam’ (pooledhuman immunoglobulin) to the other. The tubes were rotated for 1.5 h at4° C. followed by washing ×3 with 1 ml PBS each. Supernatants werediscarded. 500 μl PBS was added, mixed well and samples split evenlyinto 5× Eppendorf tubes for each sample. Tubes containing Campath andOctagam were spun and supernatants discarded. Semen samples were addedto the appropriate tubes (5× different semen samples), i.e. semen 160μl+160 μl PBS followed by rotation overnight at 4° C. Tubes were spunand supernatants collected for use in T-cell assays at 1:20 (alreadydiluted 1:2 therefore 1:10 into assay).

T-cell proliferation in response to antigen (TT) in PBMCs from healthydonors was measured by CFSE dye dilution (Mannering et al., 2003).CFSE-labelled cells (2×10⁵/well, 100 μl) were cultured in 96-well roundbottom plates in replicates of 6 with medium alone or with TT±CF1D12anti-CD52 mAb (final concentration 20 μg/ml). The latter was added ateither at 0 or 20 hr, the later time to allow initiation of activationof T-cells given that the receptor for soluble CD52, Siglec-10, wasshown to be up-regulated by activation (FIG. 12B). Unstained cells werealso included and used to set the compensations on the flow cytometer.The cell division index (CDI) was calculated as the ratio of the numberof divided CFSE^(dim)CD4⁺ cells per 20,000 undivided CFSE^(bright) CD4⁺cells in the presence of antigen to the number of divided CFSE^(dim)CD4⁺ cells per 20,000 undivided CFSE^(bright) CD4⁺ in the absence ofantigen.

Results

FIG. 21 illustrates the presence of soluble CD52 in 26 semen samplesover serial dilutions. Generally, semen contains high levels of solubleCD52 that titer out over several log dilutions.

As shown in FIG. 22, antigen (TT) alone dramatically increased T-cellproliferation (see ‘No semen’ bars in FIG. 22). However, this effect wassignificantly reduced in the presence of semen (see ‘TT’ for semensamples #1 and #15 in FIG. 22). A single round of immunodepletion ofCD52 using the anti-CD52 antibody Campath partially reversed theinhibitory effect of semen (see ‘Campath+TT’ bars for semen samples #1and #15 in FIG. 22). No significant reversal was seen with the controlIgG immunodepleted samples.

As shown in FIG. 23, antigen (TT) alone dramatically increased T-cellproliferation (see ‘No semen’ bars in FIG. 23). Addition of theanti-CD52 antibody CF1D12 further increased T-cell proliferation.However, in the presence of semen, T-cell proliferation was dramaticallyreduced (see ‘TT’ for semen samples #14, #20 and #22 in FIG. 23). Thus,semen increases unstimulated and decreased antigen-stimulatedproliferation. Addition of the anti-CD52 antibody CF1D12 partiallyreversed the inhibitory effect of semen.

Thus, semen-derived soluble CD52 achieved the same suppressive effect oneffector T-cell function (exemplified in this Example by T-cellproliferation) as lymphocyte-derived soluble CD52, demonstrating thatalternative carbohydrate moieties can be present on the solubleglycoprotein disclosed herein without diminishing its inhibitoryfunction.

Example 21 CD52-Fc Effects on Monocytes Methods and Results

THP-1 cells (human acute monocytic leukemia cell line) were grown inRPMI-1640 medium supplemented with 10% FCS, 2 mM glutamine and 50 μM2-mercaptoethanol. Cells were seeded at 2×10⁵/well in IMDM containing 5%pooled, heat-inactivated human serum, 100 mM non-essential amino acids,2 mM glutamine and 50 μM 2-mercaptoethanol (IP5 medium) at 37° C. under5% CO₂.

Cells were incubated with different doses of CD52-Fc or Fc control inpresence of LPS (100 ng/ml) for 24 hr. Medium was collected and theconcentration of IL-1β measured by ELISA. The results of this experimentare summarized in FIG. 24.

In a further experiment, cells were incubated with different doses ofCD52-Fc or Fc control in presence of the TLR-2 agonist Pam3CSK (100ng/ml) for 24 hr. Media were then collected and the concentration ofIL-1β measured by ELISA. The results of this experiment are summarizedin FIG. 25.

THP-1 cells were also differentiated for 3 hr with 500 nMphorbol-12-myristate-13-acetate (PMA). The cells were then washed andseeded at 2×10⁵/well in IP-5 medium and incubated overnight at 37° C.under 5% CO₂. The next morning the medium was changed and cells wereincubated with CD52-Fc or Fc control (50 μg/ml) in presence of alum (100μg/ml) for 16 hour. Medium was collected and the concentration of IL-1βmeasured by ELISA. The results of this experiment are summarized in FIG.26.

Bone marrow from 10 week-old C57/B6 mice was differentiated for 7 daysin granulocyte-macrophage colony stimulating factor (10 ng/ml) inKDS-RPMI medium-10% FCS. Bone marrow-derived dendritic cells (BMDCs)were collected, washed and seeded at 2×10⁴/well in a 96-well plate.Cells were incubated with 40 μg/ml mouse CD52-Fc or PBS (Control) inpresence of LPS (800 ng/ml), CPG (0.8 μM) or Listeria monocytogenes(8×106/well). In addition, cells were primed for 3 hr with LPS (100ng/ml) and then stimulated with the known inflammsome agonists,monosodium urate (MSU) (150 μg/ml), alum (150 μg/ml) and nigericin (1μM). After 24 hr, media were collected and cytokine concentrationsmeasured by multiplex cytokine array assay. The results of thisexperiment using IL-1β are summarized in FIG. 27. Similar results wereobtained for IL-1α, TNF-α, MCP-1, IL-6, IL-9 and IL-12 (data not shown).

Mouse CD52-Fc (250 μg) was incubated with neuraminidase fromArthrobacter ureafaciens (2 unit) or reaction buffer (250 mM sodiumphosphate, pH 6.0) at 37° C. overnight, and the reaction terminated byheating at 75° C. for 5 minutes. THP-1 cells were incubated withneuraminidase- or reaction buffer-treated mCD52-Fc (final 12.5 μg/ml) inpresence of LPS (100 ng/ml) for 24 hr. Media were collected and theconcentration of IL-1β measured by ELISA. The results of this experimentare summarized in FIG. 28.

Mouse CD52-Fc (300 μg) was treated with or without PNGase F under thesame conditions, according to the instructions of the manufacturer(BioLabs Inc.). Removal of N-linked oligosaccharide with reduction inthe molecular weight of CD52-Fc was confirmed by SDS-PAGE and CoomassieBlue staining. The protein solutions were then desalted by dialysisagainst pure sterile water. THP-1 cells were seeded at 2×10⁵/well in IP5medium and incubated with treated CD52-Fc (final 30 μg/ml) orglycosylated CD52-Fc in presence of 100 ng/ml LPS for 24 hr. Media werecollected and the concentration of IL-1β measured by ELISA. The resultsof this experiment are summarized in FIG. 29.

Discussion

In response to a range of inflammatory stimuli, CD52-Fc in adose-dependent manner suppressed IL-1β secretion by the human THP1monocyte line and by mouse bone marrow-derived dendritic cells.Furthermore, as shown for T cells, this suppressive effect of CD52-Fcdepends on its oligosaccharide moiety because it was abrogated by priortreatment of CD52-Fc with neuraminidase to remove terminal sialic acidsor with PNGase-F to remove the N-linked oligosaccharide itself. Thesefindings demonstrate that the suppressive effects of CD52-Fc shown for Tcells extend to other cell types that participate in innate immunityand, again similar to T cells, are presumably mediated by a Siglecreceptor.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from U.S. 61/560,254 filed 15Nov. 2011 and U.S. 61/705,633 filed 26 Sep. 2012, the entire contents ofboth of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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1.-30. (canceled)
 31. A method of treating or preventing a disease orcondition mediated by effector T-cell function, in a mammalian subject,the method comprising administering a therapeutically effective amountof any one or more of: i) soluble CD52 glycoprotein, ii) a fusionprotein comprising soluble CD52 glycoprotein as a first protein, and asecond protein; iii) a polynucleotide encoding the peptide portion ofsoluble CD52 glycoprotein of part i) or the fusion protein of part ii);iv) a vector comprising the polynucleotide of part iii); v) an isolatedcell comprising the polynucleotide of part iii) or the vector of partiv); vi) an isolated CD52^(hi) cell capable of producing soluble CD52glycoprotein; vii) an isolated cell population comprising a plurality ofCD52^(hi) cells capable of producing soluble CD52 glycoprotein; viii)cell culture medium, or a fraction thereof comprising soluble CD52glycoprotein, isolated from a cell culture comprising the cell of partvi) or the cell population of part vii); ix) an agent capable ofincreasing the level of expression of soluble CD52 glycoprotein by acell; and x) the pharmaceutical composition of comprising any one ormore of i) to ix) and a pharmaceutically acceptable carrier, to thesubject wherein the disease or condition comprises an autoimmunedisease, an allograft rejection, a graft-versus-host reaction, or anallergic disease.
 32. (canceled)
 33. (canceled)
 34. The method of claim31, wherein the disease comprises an autoimmune disease, wherein theautoimmune disease is selected from the group consisting of multiplesclerosis and rheumatoid arthritis.
 35. The method of claim 31, whereinthe condition mediated by effector T-cell function is an allograftrejection or a graft-versus-host reaction.
 36. The method of claim 31,wherein the soluble CD52 glycoprotein, fusion protein, cell culturemedium, agent or composition is administered at a mucosal or transdermalsite. 37.-47. (canceled)
 48. The method of claim 34, wherein theautoimmune disease is multiple sclerosis.
 49. The method of claim 48,wherein the method comprises administering a therapeutically effectiveamount of a soluble CD52 glycoprotein.
 50. The method of claim 49,wherein the soluble CD52 glycoprotein comprises an amino acid sequenceat least 80% identical to the amino acid sequence of GQNDTSQTSSPS (SEQID NO: 3).
 51. The method of claim 49, wherein the soluble CD52glycoprotein comprises an amino acid sequence at least 90% identical tothe amino acid sequence of GQNDTSQTSSPS (SEQ ID NO: 3).
 52. The methodof claim 49, the soluble CD52 glycoprotein comprises the amino acidsequence of GQNDTSQTSSPS (SEQ ID NO: 3).
 53. The method of claim 49,wherein the method comprises administering the soluble CD52 at aparenteral site.
 54. The method of claim 34, wherein the autoimmunedisease is rheumatoid arthritis.
 55. The method of claim 54, wherein themethod comprises administering a therapeutically effective amount of asoluble CD52 glycoprotein.
 56. The method of claim 55, wherein thesoluble CD52 glycoprotein comprises an amino acid sequence at least 80%identical to the amino acid sequence of GQNDTSQTSSPS (SEQ ID NO: 3). 57.The method of claim 55, wherein the soluble CD52 glycoprotein comprisesan amino acid sequence at least 90% identical to the amino acid sequenceof GQNDTSQTSSPS (SEQ ID NO: 3).
 58. The method of claim 55, the solubleCD52 glycoprotein comprises the amino acid sequence of GQNDTSQTSSPS (SEQID NO: 3).
 59. The method of claim 55, wherein the method comprisesadministering the soluble CD52 at a parenteral site.